Methods and compositions for inhibiting oxidative stress

ABSTRACT

The present invention is directed to methods for the treatment or prevention of oxidative stress in a cell, e.g., photoreceptor cell, and methods for the treatment and prevention of disorders associated therewith by the administration of an agent, e.g., a nucleic acid molecule, which increases the expression and/or activity of an antioxidant defense protein.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/032,426, filed on Apr. 27, 2016, which is a 35 U.S.C. § 371 nationalstage filing of International Application No. PCT/US2014/062917, filedon Oct. 29, 2014, which in turn claims the benefit of priority to U.S.Provisional Patent Application No. 61/896,805, filed on Oct. 29, 2013.The entire contents of each of the foregoing applications areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contractEY023291-01 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Cells can be compromised by genetic and environmental factors that leadto their malfunction and death. For example, in the retina, specializedsensory neurons, the photoreceptors (rods and cones), as well as retinalganglion cells (RPG), the output neurons of the retina, are the neuronalcell types that can malfunction and die due to genetic and/orenvironmental reasons, leading to partial or complete loss of vision.

The retina contains two major types of light-sensitive photoreceptorcells, i.e., rod cells and cone cells. Cone cells are responsible forcolor vision and require brighter light to function, as compared to rodcells. There are three types of cones, maximally sensitive tolong-wavelength, medium-wavelength, and short-wavelength light (oftenreferred to as red, green, and blue, respectively, though thesensitivity peaks are not actually at these colors). Cones are mostlyconcentrated in and near the fovea. Only a small percentage ofphotoreceptors are cones in the periphery of the retina. Objects areseen most sharply in focus when their images fall on the cone-enrichedspot, as when one looks at an object directly. Cone cells and rods areconnected through intermediate cells in the retina to nerve fibers ofthe optic nerve. When rods and cones are stimulated by light, the nervessend off impulses through these fibers to the brain.

Reduced viability of cone cells is associated with various retinaldisorders, in particular, retinitis pigmentosa. Retinitis pigmentosa isa family of inherited retinal degenerations (RD) that is currentlyincurable and frequently leads to blindness. Affecting roughly 1 in3,000 individuals, it is the most prevalent form of RD caused by asingle disease allele (RetNet, www.sph.uth.tmc.edu/Retnet/). Thephenotype is characterized by an initial loss of night vision due to themalfunction and death of rod photoreceptors, followed by a progressiveloss of cones (Madreperla, S. A., et al. (1990) Arch Ophthalmol 108,358-61). Additionally, retinitis pigmentosa is further characterized by,e.g., night blindness, progressive loss of peripheral vision, eventuallyleading to total blindness, ophthalmoscopic changes consisting of darkmosaic-like retinal pigmentation, attenuation of the retinal vessels,waxy pallor of the optic disc, and in the advanced forms, maculardegeneration. Since cones are responsible for color and high acuityvision, it is their loss that leads to a reduction in the quality oflife. In many cases, the disease-causing allele is expressed exclusivelyin rods; nonetheless, cone cell death follows rod cell death. Indeed, todate there is no known form of RD in humans or mice where rods die, andcones survive. In contrast, mutations in cone-specific genes result onlyin cone death.

Thus, there is a need in the art for therapies to prevent, treat,diagnose and prognose vision loss that results from decreased retinalcell function.

SUMMARY OF THE INVENTION

The present invention is directed to methods for inhibiting oxidativestress in a cell, e.g., a photoreceptor cell, as well as methods fortreating or preventing a disorder associated with oxidative stress,e.g., oxidative stress of a photoreceptor cell. The present invention isbased, at least in part, on the discovery that increased expressionand/or activity of certain genes (genes encoding antioxidant defenseproteins) in a photoreceptor cell undergoing oxidative stress can serveto fight oxidation and/or detoxify free radicals. In particular, theincreased expression of genes encoding proteins involved in generalup-regulation of the anti-oxidation program (e.g., transcriptionfactors), as well as the increased expression of genes encodingantioxidant enzymes that detoxify free radicals have been shown to,increase photoreceptor viability.

Accordingly, the present invention provides methods for inhibitingoxidative stress of a photoreceptor cell, as well as methods for thetreatment and/or prevention of disorders associated with cellularoxidative stress, for example, retinitis pigmentosa, by increasing theexpression and/or activity of an antioxidant defense protein, includinge.g., superoxide dismutase 2 (SOD2), catalase, peroxisomeproliferator-activated receptor gamma coactivator 1-alpha (PGC1α), andnuclear factor erythroid 2-like 2 (Nrf2).

In one aspect, the present invention provides a method for inhibitingoxidative stress of a photoreceptor cell compromised by a retinaldisorder. The method includes contacting the cell with an agent thatincreases the expression and/or activity of an antioxidant defenseprotein, thereby inhibiting oxidative stress of the photoreceptor cell.

In another aspect, the present invention provides a method forinhibiting oxidative stress of a cell. The method includes contactingthe cell with nucleic acid molecules that encode catalase, SOD2, PGC1α,and/or Nrf2, or any combination or sub-combinations thereof, therebyinhibiting oxidative stress in a cell. In some embodiments, the cell isa photoreceptor cell such as, e.g., cone and/or rod cell. In someembodiments, the cell is a neuronal cell. In certain embodiments, thecell is a neuronal cell, a defect in which gives rise to a neurologicaldisorder.

In another aspect, the present invention provides a method forprolonging the viability of a photoreceptor cell compromised by aretinal disorder. The method includes contacting the cell with an agentthat increases the expression and/or activity of an antioxidant defenseprotein, thereby inhibiting oxidative stress of the photoreceptor cell.In certain embodiments, the viability of a photoreceptor cell isprolonged, e.g., for about 1 week, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about3 months, about 4 months, about 5 months, about 6 months, about 7months, about 8 months, about 9 months, about 10 months, about 11months, about 12 months, about 2 years, about 3 years, about 4 years,about 5 years, about 10 years, about 15, years, about 20 years, about 25years, about 30 years, about 40 years, about 50 years, about 60 years,about 70 years, and about 80 years.

In another aspect, the present invention provides a method for treatingor preventing a retinal disorder in a subject. The method includesadministering to the subject an agent that increases the expressionand/or activity and of an antioxidant defense protein, thereby treatingor preventing the ocular disorder.

In yet another aspect, the present invention provides a method fortreating or preventing retinitis pigmentosa in a subject. The methodincludes administering to the subject an agent that increases theexpression and/or activity of an antioxidant defense protein, therebytreating or preventing retinitis pigmentosa in the subject.

In a further aspect, the present invention provides a method fortreating or preventing a disorder associated with oxidative stress in asubject. The method includes administering to the subject one or morenucleic acid molecules that encode catalase, SOD2, PGC1α, and/or Nrf2,thereby treating or preventing a disorder associated with oxidativestress in the subject. In some embodiments, the disorder is an oculardisorder. In some embodiments, the disorder is a neurological disorder,e.g., Alzheimer's Disease, Parkinson Disease, Huntington's Disease, andAmyotrophic Lateral Sclerosis.

In any of the foregoing aspects of the invention, the increasedexpression of an antioxidant defense protein reduces the level ofreactive oxygen species (ROS). In certain embodiments, ROS include,e.g., free radical species. In some embodiments, the antioxidant defenseprotein combats free radicals. In certain embodiments of the foregoingmethods, the photoreceptor cell is a cone and/or rod cell.

In some embodiments, the ocular disorder is selected from the groupconsisting of retinitis pigmentosa, age related macular degeneration,cone rod dystrophy, rod cone dystrophy, and glaucoma. In one embodiment,the ocular disorder is a retinal disorder.

In certain embodiments of the foregoing methods, the ocular disorder isassociated with decreased viability of cone cells and/or decreasedviability of rod cells.

In some embodiments, the ocular disorder is a genetic disorder. In otherembodiments, the ocular disorder is not associated with blood vesselleakage and/or growth. In certain embodiments, the ocular disorder isnot associated with diabetes and/or diabetic retinopathy. In furtherembodiments, the ocular disorder is not NARP (neuropathy, ataxia andretinitis pigmentosa).

In one embodiment of the foregoing methods, the agent is a nucleic acidmolecule encoding an antioxidant defense protein. In certainembodiments, the nucleic acid molecule comprises an antioxidant defensegene. In some embodiments, the antioxidant defense gene encodes atranscription factor or an antioxidant enzyme. In particularembodiments, the antioxidant defense gene is selected from the groupconsisting of catalase, superoxide dismutase 2 (SOD2), peroxisomeproliferator-activated receptor gamma coactivator 1-alpha (PGC1α),nuclear factor erythroid 2-like 2 (Nrf2), and any combination thereof.

Thus, any of the following combinations of antioxidant defense genes maybe used in the methods of the invention: catalase; SOD2; PGC1α; nrf2;catalase and SOD2; SOD2 and PGC1α; PGC1a and nrf2; catalase and PGC1α;catalase and nrf2; SOD2 and nrf2; catalase, SOD2, and PGC1α; SOD2,PGC1α, and nrf2; catalase, PGC1α, and nrf2; catalase, SOD2, and nrf2; orcatalase, SOD2, PGC1α, and nrf2.

In certain embodiments, the nucleic acid molecule is contained within avector. In an exemplary embodiment, the vector is selected from thegroup consisting of a retroviral vector, an adenoviral vector, anadenoviral/retroviral chimera vector, an adeno-associated virus (AAV)vector, a herpes simplex viral I or II vector, a parvovirus vector, areticuloendotheliosis virus vector, a poliovirus vector, apapillomavirus vector, a vaccinia virus vector, and a lentivirus vector.In one embodiment, the vector is an AAV vector, for example, an AAV 2/5or an AAV 2/8 vector.

In the foregoing aspects of treating or preventing a retinal disorder,or specifically retinitis pigmentosa, the administration is intraocularadministration. In exemplary embodiments, the intraocular administrationis selected from the group consisting of intravitreal, subconjunctival,sub-tenon, periocular, retrobulbar, suprachoroidal, and intrascleraladministration. In one embodiment, the intraocular administration issub-retinal or intravitreal administration.

In another aspect, the present invention provides methods for treatingor preventing retinitis pigmentosa in a subject. The methods includeadministering to the subject an isolated Nrf2 nucleic acid molecule,thereby treating or preventing retinitis pigmentosa in the subject.

In yet another aspect, the present invention provides methods fortreating or preventing retinitis pigmentosa in a subject. The methodsinclude administering to the subject an isolated Nrf2 nucleic acidmolecule and an isolated PGC1a nucleic acid molecule, thereby treatingor preventing retinitis pigmentosa in the subject.

In one embodiment, the nucleic acid molecule is contained within avector, such as a vector selected from the group consisting of aretroviral vector, an adenoviral vector, an adenoviral/retroviralchimera vector, an adeno-associated virus (AAV) vector, a herpes simplexviral I or II vector, a parvovirus vector, a reticuloendotheliosis virusvector, a poliovirus vector, a papillomavirus vector, a vaccinia virusvector, and a lentivirus vector. In one embodiment, the vector is an AAVvector, e.g., an AAV 2/5 or an AAV 2/8 vector.

In one embodiment, the administration is intraocular administration,such as intravitreal, sub-retinal, subconjunctival, sub-tenon,periocular, retrobulbar, suprachoroidal, and intrascleraladministration. In one particular embodiment, the intraocularadministration is sub-retinal or intravitreal administration.

In one aspect, the present invention provides pharmaceuticalcompositions suitable for intraocular administration, comprising anisolated Nrf2 nucleic acid molecule.

In another aspect, the present invention provides pharmaceuticalcompositions suitable for intraocular administration, comprising anisolated Nrf2 nucleic acid molecule and an isolated PGC1a nucleic acidmolecule.

In one embodiment, a therapeutically or prophylactically effectiveamount of the nucleic acid molecule is contained in the compositionssuitable for intraocular administration.

In one aspect, the present invention provides compositions comprising aviral vector comprising a retinal cell-type specific promoter operablylinked to a nucleic acid molecule encoding Nrf2.

In another aspect, the present invention provides compositionscomprising a viral vector comprising a retinal cell-type specificpromoter operably linked to a nucleic acid molecule encoding PGC1α.

In yet another aspect, the present invention provides compositionscomprising a viral vector comprising a retinal cell-type specificpromoter operably linked to a nucleic acid molecule encoding Nrf2 andPGC1α.

The retinal cell-type specific promoter may be a rod-specific promoter,a cone-specific promoter, and/or a rod- and cone-specific promoter.

In one embodiment, the composition is suitable for intraocularadministration, e.g., sub-retinal or intravitreal administration.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict the infection of WT and rd1 retinas with AAV-CMV-GFP.

FIG. 1A is a low magnification image of a cryosection of a WT(wild-type) retina infected at P0 with AAVCMV-GFP, and harvested at P30showing extensive spread of the infection throughout the retina.

FIG. 1B is a higher magnification view of the outer nuclear layer inFIG. 1A, showing that cones are well infected and express GFP at a highlevel (anti-GFP in light gray, PNA—a cone marker—in medium gray).Bracket indicates entirety of cones, with top arrow pointing to coneouter segment (OS) (medium gray PNA stain), middle arrow to cone innersegment (IS) and bottom arrow to cone cell body.

FIG. 1C is a low magnification image of a cryosection of an rd1 retinainfected at P0 with AAVCMV-GFP, and harvested at P30.

FIG. 1D is a higher magnification view of FIG. 1C, with remaining conesat P30 well stained with anti-GFP (light gray) and PNA (medium gray).Arrow points to a single cone, and bracket indicates layer of cone cellbodies.

FIG. 1E is an image of the rods from the retina shown in FIG. 1A,visualized by a longer exposure to show that they are infected butexpress at a lower level (compare to FIG. 1B, with arrow pointing to acone).

FIG. 1F is a flat mount image of an rd1 retina infected with AAV-CMV-GFPat P0. A 60× (250 micron square area) image taken 1 mm from the opticnerve head, with the focal plane in the remaining outer nuclear layer isshown, to represent the type of quantification that was carried out inretinas infected with experimental viruses.

FIGS. 2A-2D depict endogenous expression of anti-oxidation enzymes inthe WT retina. Antisera was applied to cryosections of WT P30 retinas(medium gray) and anti-PNA was used to identify cone OS (light gray).

FIG. 2A is a cryosection of a WT P30 retina stained with anti-SOD2 andanti-PNA.

FIG. 2B is a cryosection of a WT P30 retina stained with anti-SOD2 tohighlight staining in cone OS. Arrows indicate cones.

FIG. 2C is a cryosection of a WT P30 retina stained with anti-Gpx1 andanti-PNA.

FIG. 2D is a cryosection of a WT P30 retina stained with anti-Gpx1 only.Arrows indicate cones. Upper bracket denotes area of OS and lowerbracket area of cone cell bodies and IS.

FIGS. 3A-3D depict that knock-down of SOD2 generates oxidation products.An shRNA directed to SOD2, or an irrelevant shRNA, was delivered byelectroporation at P0 into WT retinas. A co-electroporated plasmid foridentification of electroporated cells was included (CAG-GFP).

FIG. 3A is a cryosection from retina at P30 electroporated with controlshRNA, stained with anti-GFP (light gray), and for acrolein, anindicator of oxidized lipids (medium gray).

FIG. 3B is a cryosection from retina at P30 electroporated with controlshRNA, stained with acrolein, and no cells were positive.

FIG. 3C is a cryosection from retina at P30 electroporated with SOD2shRNA, stained with anti-GFP and acrolein.

FIG. 3D is a cryosection from retina at P30 electroporated with SOD2shRNA, stained with acrolein. All acrolein positive cells were GFP+. Twoexamples are indicated by arrows. Upper bracket denotes OS and IS, lowerbracket the outer nuclear layer (cell bodies of rods and cones).

FIGS. 4A-4C depict that knock-down of Gpx1 leads to rapid cell death. AnshRNA directed to Gpx1 was delivered by electroporation at P0 into WTmice. A co-electroporated plasmid for identification of electroporatedcells was included (CAG-GFP).

FIG. 4A is a low magnification cryosection from retina at P30 stainedwith DAPI (medium gray area), with electroporated region indicated bythe arrow.

FIG. 4B is a low magnification cryosection from retina at P30 stainedwith TUNEL. (B) TUNEL staining revealed positive cells in the area ofelectroporation.

FIG. 4C is a low magnification cryosection from retina at P30 stainedwith anti-GFP. Anti-GFP shows area that was electroporated is the areawith TUNEL+ cells. Control shRNA showed very few TUNEL+ cells (notshown).

FIGS. 5A-5C-depict endogenous expression of PGC1α in the retina. A WTadult retina was stained with anti-PGC1α. Most nuclei were positive,with higher expression in cones than in rods.

FIG. 5A is an image of a WT adult retina stained with anti-PGC1α.Anti-PGC1α in medium gray showing nuclear localization in most retinalcells (arrow in panel A, and circular medium gray staining). Arrow showsa cone nucleus.

FIG. 5B is the same section as FIG. 5A but showing expression in conesusing anti-PNA (light gray, e.g., light vertical lines at top of imageand nuclei (dark gray, DAPI, e.g., the large area in which the arrowlies). Arrow shows a cone nucleus.

FIG. 5C is an image of a WT adult retina stained with anti-cone arrestin(light gray) and anti-PGC1α showing cone staining as well. Arrow showsan example of a double stained cone.

FIGS. 6A-6C depict endogenous expression of Nrf2 in the WT, rd10 andrho−/− retinas. Antisera of Nrf2 protein was applied to cryosections ofWT P30 retinas (medium gray) and anti PNA was used to identify cone OS(light gray).

FIG. 6A is an image of a cryosection of a WT P30 retina stained withanti-Nrf2. Low level of Nrf2 (gray) is expressed in IS, outer nuclearlayer (ONL) and inner nuclear layer (INL) in WT retina.

FIG. 6B is an image of a cryosection of an rd10 P18 retina stained withanti-Nrf2. Nrf2 level (medium gray) is elevated in cones (higher thanrods) in rd10 P18 retinas. Arrows point out the cone somas with higherlevel of Nrf2 protein. Brackets denote ONL layer.

FIG. 6C is an image of a cryosection of a rho−/− P30 retina stained withanti-Nrf2. Nrf2 level (medium gray) is elevated in cones (higher thanrods) in rho−/− P30 retinas. Arrows point out the cone somas with higherlevel of Nrf2 protein. Brackets denote ONL layer.

FIG. 7 depicts that co-injection with high titer AAV vectors leads tomany cells being co-infected. A P0 WT retina was injected with a mixtureof two AAV vectors, one encoding GFP (light gray) and one encodingtdTomato (bright light gray). Arrow indicates an example of a doubleinfected cone. Quantification of 200 cells across 2 retinas showed that100% of cones, identified by PNA staining, were co-infected and 85% ofall photoreceptors were co-infected.

FIG. 8 provides schematics of AAV2/8 vector genomes used for infections.They include a CMV promoter and a beta-globin intron constructed withanti-oxidation genes. To allow for tracking of infected cells,AAV-CMV-GFP was mixed with AAV vectors expressing an antioxidant gene.

FIGS. 9A-9D and FIGS. 9A′-9D′ depict expression of antioxidant enzymesSOD2 and Catalase by AAV vectors in rd1 retinas.

FIG. 9A is an image of a cryosection of a control rd1 retina withAAV-CMV-GFP infection stained for GFP and SOD2.

FIG. 9B is an image of a cryosection of an rd1 retina with GFP, SOD2,and Catalase infection stained for GFP and SOD2.

FIG. 9C is an image of a cryosection of a control rd1 retina withAAV-CMV-GFP infection stained for GFP and Catalase.

FIG. 9D is an image of a cryosection of an rd1 retina with GFP, SOD2,and Catalase infection stained for GFP and Catalase.

FIG. 9A′ is an image of a cryosection of a control rd1 retina withAAV-CMV-GFP infection stained for SOD2.

FIG. 9B′ is an image of a cryosection of an rd1 retina with GFP, SOD2,and Catalase infection stained for SOD2.

FIG. 9C′ is an image of a cryosection of a control rd1 retina withAAV-CMV-GFP infection stained for Catalase.

FIG. 9D′ is an image of a cryosection of an rd1 retina with GFP, SOD2,and Catalase infection stained for Catalase.

Antisera of antioxidant enzymes (medium gray, FIGS. 9A′, 9B′, 9C′, and9D′) was applied to cryosections of rd1 P60 retinas and GFP expression(light gray, FIGS. 9A, 9B, 9C, and 9D) was used to track remainingcones. Control rd1 retinas with only AAV-CMV-GFP infection showed littleSOD2 (FIGS. 9A and 9A′) and Catalase (FIGS. 9C and 9C′) expression,while high level of SOD2 (FIGS. 9B and 9B′) and Catalase (FIGS. 9Daaaand 9D′) expression was evident in the retinas infected with the mixtureof three viruses.

FIGS. 10A-10D depict that delivery of anti-oxidation enzyme genes by AAVpromotes cone survival.

FIG. 10A is an image of a section through the central retina of controlrd1 retina infected with AAV-CMV-GFP.

FIG. 10B is an image of a section through the central retina of controlrd1 retina infected with AAV-CMV-GFP.

FIG. 10C is an image of a section through the central retina of an rd1mouse co-infected sub-retinally with AAV-CMV-GFP, AAV-CMV-SOD2- andAAV-CMV-catalase (vectors shown in FIG. 8).

FIG. 10D is an image of a section through the central retina of an rd1mouse co-infected sub-retinally with AAV-CMV-GFP, AAV-CMV-SOD2- andAAV-CMV-catalase (vectors shown in FIG. 8).

Retinas were infected at P0. At P50, cryosections were prepared andsections through the central retinas were imaged for GFP (light gray,FIG. 10B, and FIG. 10D). Retinal section infected with control virus(FIG. 10A, and FIG. 10B). Note absence of GFP+ cells in the centralretina (FIG. 10B). Retinal section infected with anti-oxidation viruses(FIG. 10C, and FIG. 10D). Note that there are GFP+ cells in the centralretina (arrow, FIG. 10D), in the scleral location, indicative of conecell bodies (e.g., see FIG. 1D).

FIG. 11 demonstrates that overexpression SOD2 and Catalase prolongs conesurvival. The average cone densities in WT and rd1 retinas infected withAAV vectors expressing SOD2 and Catalase were shown. Cone density wasquantified in four 250 μm×250 μm squares at 1.5 mm dorsal, ventral,nasal and temporal to the center of the optic nerve head in each retina,and around 20 retinas were analyzed per group per time point. Adult WTretinas have a constant cone density (˜350 cones per 0.0625 mm²). Themean (±SEM) cone density was greater at P50, P60 and P70 and wassignificantly greater (p<0.05) at P50 in the rd1 retinas treated withAAV-CMV-GFP, AAV-CMV-SOD2 and AAV-CMV-Catalase (medium gray line)compared to the control retinas with only AAV-CMV-GFP (dark gray line).

FIGS. 12A-12C, FIGS. 12A′-12C′, and FIG. 12D demonstrate thatoverexpression of SOD2 and Catalase preserves outer segments (OS) andinner segments (IS) of cones.

FIG. 12A is a high magnitude image of a wild-type retina stained withanti PNA to show cone OS and IS (medium gray).

FIG. 12B is a high magnitude image of an rd1 retina infected with anAAV-GFP vector. Retinas were stained with GFP anti PNA to show cone OSand IS (medium gray).

FIG. 12B′ is a high magnitude image of an rd1 retina infected with anAAV-GFP vector. Retinas were stained with anti PNA to show cone OS andIS (medium gray).

FIG. 12C is a high magnitude image of an rd1 retina infected with AAVvectors expressing SOD2 and Catalase. Retinas were stained with GFP andanti PNA (medium gray) to show cone OS and IS.

FIG. 12C′ is a high magnitude image of an rd1 retina infected with AAVvectors expressing SOD2 and Catalase. Retinas were stained with anti PNA(medium gray) to show cone OS and IS.

While long OS and IS (FIG. 12A) were present in WT retina, the remainingcones (marked by GFP, light gray, FIG. 12B) in rd1 P60 retina lack OSand IS (FIG. 12B′). Overexpression of SOD2 and Catalase rescued cone OSand IS (FIG. 12C and FIG. 12C′) in rd1 P60 retinas.

FIG. 12D is a graph depicting the percent of cone with outer sequencesin rd1 retinas infected with AAV GFP and in rd1 retinas infected withAAV vectors expressing SOD2 and Catalase. The phenotype was quantifiedby counting the percentage of cones with PNA staining. Numbers wereshown as mean±s.d. n=3 retinas per group.

FIGS. 13A-13B demonstrate that overexpression of SOD2 and Catalasepreserves photoreceptor function as measured by optomotor assay.

FIG. 13A is a graph showing the visual acuity of uninfected left eyes(medium gray) and AAV vectors injected right eyes (light gray) of rd10mice. Control treated mice with AAV-CMV-GFP (mouse ID 1-5, n=5) andAAV-CMV-GFP+AAV-CMV-SOD2+AAV-CMV-Catalase treated mice (mouse ID 6-10,n=5) were tested at P40.

FIG. 13B is a graph showing the visual acuity of uninfected left eyes(medium gray) and AAV vectors injected right eyes (light gray) of rd10mice. Control treated mice with AAV-CMV-GFP (mouse ID 1-5, n=5) andAAV-CMV-GFP+AAV-CMV-SOD2+AAV-CMV-Catalase treated mice (mouse ID 6-10,n=5) were tested at P50.

FIGS. 13A and 13B demonstrate that, at P40 and P50, most antioxidanttreated mice tested had higher right eye visual acuity than left, whilemost control treated mice have similar left and right eye response. Thedifferences of left and right eyes between the two groups (controltreated vs antioxidant AAV treated mice) were significant at P50(P<0.05) (B).

FIG. 14 demonstrates that overexpression of SOD2 and Catalase preservesphotoreceptor function as measured by light-evoked ganglion cellactivity. The left side of the graph shows the results of retinas fromrd10 mice treated with AAV-CMV-GFP andAAV-CMV-GFP+AAV-CMV-SOD2+AAV-CMV-Catalase that were harvested at P70 forganglion cell activity recording. Data presented are from a total of 35ganglion cells from 3 control treated retinas and a total of 26 cellsfrom 3 antioxidant AAV vector treated retinas.

The right side of the graph shows the results from retinas injected withAAV-CMV-GFP+AAV-CMV-Nrf2 of rho−/− mice that were harvested at P100 forthis experiment. Ten ganglion cells from each retina were measured forlight-evoked activity. Peak firing rate (spikes/sec) of each ganglioncell was averaged over 20 trials of 1 second light stimulus (wavelength:356 nm+505 nm, light intensity: 1010 photons cm−2 s−1).

ON, OFF and ON/OFF ganglion cells were included for this analysis. Theaverage peaking firing rate (white line) of antioxidant AAV vectorstreated retinas was higher compared to the control treated retinas, andthe difference is statistically significant (p<0.05).

FIGS. 15A-15B are photomicrographs demonstrating that overexpression ofPGC1α and Nrf2 saves cone photoreceptors in central and middle retinas.

FIG. 15A is a representative flat-mounted rd1 P50 retina from controlgroup (AAV-CMV-GFP treated).

FIG. 15B is a representative flat-mounted rd1 P50 retina fromantioxidant transcription factor group(AAV-CMV-GFP+AAV-CMV-PGC1α+AAV-CMV-Nrf2 treated).

In FIG. 15A and FIG. 15B cone photoreceptors were tracked by GFP. Theboxes to the right of A and B are magnifications of an area 1.5 mmdorsal to the optic nerve head. Note the absence of GFP in the centraland mid retina (highlighted by square) of the control animal in FIG.15A. Many GFP positive cells are evident in the antioxidanttranscription factor AAV treated retina in FIG. 15A.

FIG. 16 demonstrates that overexpression of PGC1α and Nrf2 prolongs conesurvival. The mean (±SEM) cone density of rd1 retinas treated withAAV-CMV-GFP+AAV-CMV-PGC1α+AAV-CMV-Nrf2 vectors at P30, P50 and P80 areshown (light gray line). The mean (±SEM) cone density was significantlygreater (p<0.001) at P50 in the retinas overexpressing SOD2 and catalasecompared to the control retinas with AAV-CMV-GFP. The mean cone densityof PGC1α and Nrf2 treated retinas was significantly greater than that ofcontrol retinas (p<0.001) and that of SOD2 and catalase treated retinas(p<0.001) at P50, and it remained significantly higher than that of thecontrol retinas (p<0.05) at P80.

FIGS. 17A, 17A′, 17B, and 17B′ demonstrate that overexpression of PGC1αand Nrf2 preserves outer segments (OS) and inner segments (IS) of conesin central retina.

FIG. 17A is a high magnification image of the mid dorsal region offlat-mounted P30 WT retina infected with AAV vectors expressing PGC1αand Nrf2 stained with anti-GFP and anti-PNA.

FIG. 17A′ is a high magnification image of the mid dorsal region offlat-mounted rd1 retina infected with AAV vectors expressing PGC1α andNrf2 stained with anti-PNA.

FIG. 17B is a high magnification image of the mid dorsal region offlat-mounted P30 WT retina infected with a control AAV vector stainedwith anti-GFP and anti-PNA.

FIG. 17B′ is a high magnification image of the mid dorsal region offlat-mounted P30 WT retina infected with a control AAV vector stainedwith anti-PNA.

Retinas were stained with anti PNA (FIG. 17A′ and FIG. 17B′) to showcone outer segments (OS) and inner segments (IS), with more OS and ISpresent in the retinas treated with PGC1α and Nrf2 overexpression (FIG.17A and FIG. 17A′), control rd1 retina lacked OS and IS ((FIG. 17B andFIG. 17B′).

FIGS. 18A-18D demonstrate that overexpression of Nrf2 and PGC1αpreserves cone outer segments (OS).

FIG. 18A is a photomicrograph of outer segments infected withAAV-GFP+NRF2+PGC1α stained for GFP and red/green opsin.

FIG. 18B is a photomicrograph of outer segments infected withAAV-GFP+NRF2+PGC1α stained for red/green opsin.

FIG. 18C is a photomicrograph of outer segments infected with AAV-GFPstained for GFP and red/green opsin.

FIG. 18D is a photomicrograph of outer segments infected with AAV-GFPstained for red/green opsin.

As shown in FIG. 18A and FIG. 18B, red/green opsin protein localized tothe remaining cone outer segment structure, while it mislocalized to thecytoplasm of cell soma in the control retina. Arrow indicates an exampleof the cone outer segment.

FIG. 19 is a graph demonstrating that overexpression of Nrf2 aloneprolongs cone survival. The average cone densities in rd1 retinasinfected with AAV vectors expressing GFP+PGC1α (lower line), GFP+Nrf2(upper line) and GFP+PGC1α+Nrf2 (middle line) are shown. The mean conedensity was greater in the retinas treated with AAV-CMV-GFP+AAV-CMV-Nrf2than that in those treated with AAV vectors expressingAAV-CMV-GFP+AAV-CMV-PGC1α+AAV-CMV-Nrf2 at P50.

FIG. 20 is a graph demonstrating that overexpression of Nrf2 preservescone outer segments. The phenotype was quantified by counting thepercentage of cones with PNA staining. The mid dorsal regions of the P50retinas were chosen for quantification. Numbers are shown as mean±SEM.

FIGS. 21A-21B are photomicrographs demonstrating that treatment of Nrf2reduces superoxide levels. Hydroethidine (DHE) was injectedintraperitoneally at postnatal day (P) 45 into rd10 mice treated withAAV-CMV-GFP or rd10 mice treated with AAV-CMV-GFP+AAV-CMV-Nrf2. After 18hours, mice were euthanized and retinas were harvested.

FIG. 21A is a photomicrograph of a retina from a rd10 P45 mouse treatedwith AAV-CMV-GFP+AAV-CMV-Nrf2.

FIG. 21B is a photomicrograph of a retina from a rd10 P45 mouse treatedwith AAV-CMV-GFP+AAV-CMV-Nrf2.

FIG. 21C is a photomicrograph of a retina from a rd10 P45 mouse treatedwith AAV-CMV-GFP.

FIG. 21D is a photomicrograph of a retina from a rd10 P45 mouse treatedwith AAV-CMV-GFP.

Stronger fluorescence in AAV-CMV-GFP injected retinas (FIG. 21C)demonstrates higher level of superoxide species produced. Minimalfluorescence was present in the retinas treated withAAV-CMV-GFP+AAV-CMV-Nrf2 (FIG. 21A). FIG. 21B and FIG. 21D show the GFPgreen fluorescence of the retinas in FIG. 21A and FIG. 21C.

FIGS. 22A-22F demonstrates that that overexpression of Nrf2 reduceslipid oxidation in cones. P30 rd1 retinas were harvested andimmunostained against acrolein-modified proteins. Acrolein is ametabolite of lipid peroxidation and can react with proteins.

FIGS. 22A-22C are photomicrographs of control P30 rd1 retinas infectedwith AAV-CMV-GFP and immunostained against acrolein-modified proteins.

FIGS. 22D-22F are photomicrographs of P30 rd1 retinas infected withAAV-CMV-GFP+AAV-CMV-Nrf2 and immunostained against acrolein-modifiedproteins.

Retinas infected with AAV-CMV-GFP+AAV-CMV-Nrf2 (FIGS. 22D-22F) havereduced level of anti-acrolein staining, demonstrating lower levels oflipid oxidation, compared to the control retinas infected withAAV-CMV-GFP (FIGS. 22A-22C).

FIGS. 23A-23B are graphs demonstrating that overexpression of Nrf2provides for better visual acuity as shown by the optomotor assay. Theright eye of rd10 mice received AAV vector injection, while the left eyewas uninjected and served as within-animal control. The right and lefteyes of P50 rd10 mice were tested separately.

FIG. 23A is a graph showing the visual acuities of the tested eyes. Themean visual acuity (±SEM) of the right eyes which receivedAAV-CMV-GFP+AAV-CMV-Nrf2 treatment is higher than that of the right eyeswhich received AAV-CMV-GFP treatment.

FIG. 23B is a graph showing the-ratio of Right eye/Left eye visualacuity of each animal. The ratio was used to minimize the variationbetween animals without treatment. The R/L, ratio ofAAV-CMV-GFP+AAV-CMV-Nrf2 treated retinas was significantly higher thanthat of AAV-CMV-GFP treated retinas (p<0.05) (B).

FIGS. 24A-24C demonstrate that overexpression of Nrf2 results in bettervisual function as assessed by photopic electroretinography (ERG).

FIG. 24A are representative waveform graphs (treated right eye anduntreated left eye) of P40 rd10 mice tested for cone-initiatedelectrical signal by photopic ERG following treatment with anAAV-CMG-GFP+AAV-CMV-Nrf2. The right eye of an AAV-CMG-GFP+AAV-CMV-Nrf2treated mouse had a substantially better waveform.

FIG. 24B are representative waveform graphs (treated right eye anduntreated left eye) of P40 rd10 mice tested for cone-initiatedelectrical signal by photopic ERG following treatment with a controlAAV-GFP. The right eye of an AAV-CMG-GFP+AAV-CMV-Nrf2 treated mouse hada substantially better waveform.

FIG. 24C is a graph demonstrating that the ratio of right eye/left eyeb-wave amplitude was significantly higher in AAV-CMV-GFP+AAV-CMV-Nrf2treated mice than that in the control mice.

FIGS. 25A-25B demonstrate that overexpression of SOD2 and Catalasepreserves outer segments of cones in retinas of rhodopsin null mice(rho−/−). The retinas of rho−/− mice at 6 months of age were infectedwith AAV vectors expressing SOD2 and Catalase. Retinas were stained withanti PNA to show cone outer segments (OS).

FIG. 25A is an image of a retina of a rho−/− mouse at 6 months of ageinfected with AAV vectors expressing SOD2 and Catalase showing PNA andGFP staining.

FIG. 25B is an image of a retina of a rho−/− mouse at 6 months of ageinfected with AAV vectors expressing SOD2 and Catalase showing PNAstaining.

FIG. 26A is a graph depicting the effect of increased expression of Nrf2and Sod2 on retinal ganglion cell survival following optic nerve crushin wild-type mice.

FIG. 26B is a graph depicting the effect of increased expression of Nrf2and Sod2 on axon regeneration following optic nerve crush in wild-typemice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatincreased expression and/or activity of genes encoding antioxidantdefense protein(s), in a photoreceptor cell undergoing oxidative stress,can inhibit oxidative stress in the affected cell. As rods are the majorcell type in the outer nuclear layer (ONL), cones experience a greatlyaltered environment following rod death. The cone outer segment (OS)collapses, they lose their intimate association with the retinalpigmented epithelium (RPE), and are, thus, exposed to a hyperoxicenvironment as evidenced by greater oxidation of their nucleic acids,proteins, and lipids. Thus, it has been discovered that cone cells in amouse model of RP show signs of oxidation. Accordingly, increasedexpression and/or activity of an antioxidant defense protein, includingantioxidant enzymes and transcription factors that generally up-regulatethe anti-oxidation program can serve to inhibit cellular oxidativestress, thus, increasing photoreceptor viability. Accordingly, thepresent invention provides methods for inhibiting oxidative stress of aphotoreceptor, as well as methods for the treatment and/or prevention ofdisorders associated with cellular oxidative stress, for example,retinitis pigmentosa, by increasing the expression and/or activity of atleast one antioxidant defense protein.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, the terms “inhibit oxidative stress” and “inhibition ofoxidative stress” refer to preventing as well as ameliorating,decreasing, or reducing oxidative stress.

As used herein, the term “antioxidant defense protein” includes anyprotein that inhibits the oxidation of a molecule. More specifically, an“antioxidant defense protein” encompasses any upstream element (e.g.,transcription factor) involved in the anti-oxidation program, as well asany downstream element (e.g., antioxidant enzymes) that can be activatedas a result of the enhancement of an upstream element. Exemplaryantioxidant defense proteins include, superoxide dismutase 2 (SOD2),catalase, peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC1α), and nuclear factor erythroid 2-like 2 (Nrf2).

As used herein, the term “oxidative stress” refers to any imbalance inthe normal reduction-oxidation state in a photoreceptor cell that leadsto an increase in the level of reactive oxygen species (ROS) resultingin cellular damage including, for example, DNA damage, lipidperoxidation, and oxidation of proteins.

As used herein, the terms “antioxidant enzyme” and “enzymatic scavenger”are used interchangeably to refer to antioxidant proteins capable ofdetoxifying or ameliorating free radicals, i.e., reactive oxygen species(ROS). Examples include, but are not limited to, alpha-1-microglobulin,superoxide dismutases, catalases, lactoperoxidases, glutathioneperoxidases, and peroxiredoxins.

As used herein, the term “reduces the level of ROS” refers to decreasingthe level of ROS to physiologically acceptable non-toxic levels, as wellas preventing the further generation of ROS.

In one embodiment of the invention, cells suitable for use in theinstant methods are photoreceptor cells, i.e., a specialized cell foundin the retina. The retina is a thin, transparent tissue containing about120 million separate rod cells (night vision) and 7 million cone cells(day and color vision) as well as millions of other structuralsupporting and interconnecting cells. Photoreceptor cells consist of“rods” and “cones”, which are the photosensitive cells of the retina.The rods contain rhodopsin, the rod photopigment, and the cones containother distinct photopigments, which respond to light and ultimatelytrigger a neural discharge in the output cells of the retina, theganglion cells. Ultimately, this signal is registered as a visualstimulus in the visual cortex and other target locations in the brain.The retinal pigment epithelial (RPE) cells produce, store and transporta variety of factors that are responsible for the normal function andsurvival of photoreceptors. Retinal cells that can also sense lightconsist of photosensitive ganglion cells. These cells, known as themelanopsin ganglion cells are found in the inner retina, have dendritesand long axons projecting to the protectum (midbrain), thesuprachiasmatic nucleus in the hypothalamus, and the lateral geniculate(thalamus). In one embodiment, a photoreceptor cell is a rod. In someembodiments, a photoreceptor cell is distinct from the optic nerve. Inone embodiment, a photoreceptor cell is a cone. In one embodiment, aphotosensitive cell is a cell is a melanopsin ganglion cell.

As used herein, the term “retinal disorder” refers generally to adisorder of the retina. In one embodiment, the retinal disorder isassociated with oxidative stress, decreased viability, for example,death, of cone cells, and/or rod cells. Moreover, in a particularembodiment, a retinal disorder is not associated with blood vesselleakage and/or growth, for example, as is the case with diabeticretinopathy, but, instead is characterized primarily by reducedviability of cone cells and/or rod cells. In certain embodiments, theretinal disorder is a genetic disorder. In a particular embodiment, theretinal disorder is retinitis pigmentosa. In another embodiment, theretinal disorder is age-related macular degeneration. In anotherembodiment, the retinal disorder is cone-rod dystrophy. In anotherembodiment, the retinal disorder is rod-cone dystrophy. In otherembodiments, the retinal disorder is not associated with blood vesselleakage and/or growth. In certain embodiments, the retinal disorder isnot associated with diabetes and/or diabetic retinopathy. In furtherembodiments, the retinal disorder is not NARP (neuropathy, ataxia, andretinitis pigmentosa). In yet further embodiments, the retinal disorderis not a neurological disorder. In certain embodiments, the retinaldisorder is not a disorder that is associated with a compromised opticnerve and/or disorders of the brain. In the foregoing embodiments, theretinal disorder is associated with a compromised photoreceptor cell,and is not a neurological disorder.

As used herein, the term “retinitis pigmentosa” or “RP” is known in theart and encompasses a disparate group of genetic disorders of rods andcones. Retinitis pigmentosa generally refers to retinal degenerationoften characterized by the following manifestations: night blindness,progressive loss of peripheral vision, eventually leading to totalblindness; ophthalmoscopic changes consist in dark mosaic-like retinalpigmentation, attenuation of the retinal vessels, waxy pallor of theoptic disc, and in the advanced forms, macular degeneration. In somecases there can be a lack of pigmentation. Retinitis pigmentosa can beassociated to degenerative opacity of the vitreous body, and cataract.Family history is prominent in retinitis pigmentosa; the pattern ofinheritance may be autosomal recessive, autosomal dominant, or X-linked;the autosomal recessive form is the most common and can occursporadically.

As used herein, the terms “Cone-Rod Dystrophy” or “CRD” and “Rod-ConeDystrophy” or “RCD” refer to art recognized inherited progressivediseases that cause deterioration of the cone and rod photoreceptorcells and often result in blindness. CRD is characterized by reducedviability or death of cone cells followed by reduced viability or deathof rod cells. By contrast, RCD is characterized by reduced viability ordeath of rod cells followed by reduced viability or death of cone cells.

As used herein, the term “age-related macular degeneration” alsoreferred to as “macular degeneration” or “AMD”, refers to the artrecognized pathological condition which causes blindness amongst elderlyindividuals. Age related macular degeneration includes both wet and dryforms of ARMD. The dry form of ARMD, which accounts for about 90 percentof all cases, is also known as atrophic, nonexudative, or drusenoid(age-related) macular degeneration. With the dry form of ARMD, drusentypically accumulate in the retinal pigment epithelium (RPE) tissuebeneath/within the Bruch's membrane. Vision loss can then occur whendrusen interfere with the function of photoreceptors in the macula. Thedry form of ARMD results in the gradual loss of vision over many years.The dry form of ARMD can lead to the wet form of ARMD. The wet form ofARMD, also known as exudative or neovascular (age-related) maculardegeneration, can progress rapidly and cause severe damage to centralvision. The macular dystrophies include Stargardt Disease, also known asStargardt Macular Dystrophy or Fundus Flavimaculatus, which is the mostfrequently encountered juvenile onset form of macular dystrophy.

As used herein, the term “neurological disorder” encompassesneurodegenerative disorders and demyelinating disorders. In someembodiments, the compositions of this invention are useful for thetreatment of neurological disorders including but not limited toParkinson's Disease, Tauopathies, Alzheimer's Disease, DiffuseNeurofibrillary Tangles with Calcification, Supranuclear Palsy,Progressive, TDP-43 Proteinopathies, Amyotrophic Lateral Sclerosis,Multiple Sclerosis, Frontotemporai Lobar Degeneration, Lewy BodyDisease, AIDS Dementia Complex, Aphasia, Primary Progressive, PrimaryProgressive Nonfluent Aphasia, Dementia, Vascular, CADASIL, Dementia,Multi-Infarct, Diffuse Neurofibrillary Tangles with Calcification,Frontotemporai Lobar Degeneration, Frontotemporai Dementia, PrimaryProgressive Nonfluent Aphasia, Kluver-Bucy Syndrome, Pick's Disease,Motor Neuron Disease, Bulbar Palsy, Progressive, Muscular Atrophy,Spinal, Multiple System Atrophy, Olivopontocerebellar Atrophies,Shy-Drager Syndrome, Striatonigrai Degeneration, OlivopontocerebellarAtrophies, Paraneoplastic Syndromes, Nervous System, Lambert-EatonMyasthenic Syndrome, Limbic Encephalitis, Myelitis, Transverse,Opsoclonus-Myoclonus Syndrome, Paraneoplastic Cerebellar Degeneration,Paraneoplastic Polyneuropathy, Postpoliomyelitis Syndrome, PrionDiseases, Encephalopathy, Bovine Spongiform,Gerstmann-Straussler-Scheinker Disease, Insomnia, Fatal Familial, Kuru,Scrapie, Wasting Disease, Chronic, Creutzfeidt-Jakob Syndrome,Shy-Drager Syndrome, Subacute Combined Degeneration, HeredodegenerativeDisorders, Nervous System, Alexander Disease, Amyloid Neuropathies,Familial, Bulbo-Spinal Atrophy, X-Linked, Canavan Disease, CockayneSyndrome, Dystonia Musculorum Deformans, Gerstmann-Straussier-ScheinkerDisease, Hepatolenticular Degeneration, Hereditary Central NervousSystem Demyelinating Diseases, Hereditary Sensory and AutonomicNeuropathies, Hereditary Sensory and Motor Neuropathy, HuntingtonDisease, Lafora Disease, Lesch-Nyhan Syndrome, Menkes Kinky HairSyndrome, Myotonia Congenita, Myotonic Dystrophy, Neurofibromatoses,Neuronal Ceroid-Lipofuscinoses, Optic Atrophies, Hereditary,Pantothenate Kinase-Associated Neurodegeneration, Rett Syndrome, SpinalMuscular Atrophies of Childhood, Spinocerebellar Degenerations, TouretteSyndrome, Tuberous Sclerosis, Unverricht-Lundborg Syndrome, and thesimilar, and for the treatment of Alzheimer's Disease and associateddementias. In one embodiment, the neurological disease is notParkinson's Disease.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. A nucleic acid molecule used in themethods of the present invention can be isolated using standardmolecular biology techniques. Using all or portion of a nucleic acidsequence of interest as a hybridization probe, nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. For example, with regards to genomic DNA, the term“isolated” includes nucleic acid molecules which are separated from thechromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid molecule is free of sequenceswhich naturally flank the nucleic acid molecule (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid molecule) in the genomic DNAof the organism from which the nucleic acid molecule is derived.

A nucleic acid molecule for use in the methods of the invention can alsobe isolated by the polymerase chain reaction (PCR) using syntheticoligonucleotide primers designed based upon the sequence of a nucleicacid molecule of interest. A nucleic acid molecule used in the methodsof the invention can be amplified using cDNA, mRNA or, alternatively,genomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. Furthermore,oligonucleotides corresponding to nucleotide sequences of interest canbe prepared by standard synthetic techniques, e.g., using an automatedDNA synthesizer.

The nucleic acids for use in the methods of the invention can also beprepared, e.g., by standard recombinant DNA techniques. A nucleic acidof the invention can also be chemically synthesized using standardtechniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis whichhas been automated in commercially available DNA synthesizers (See e.g.,Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No.4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,incorporated by reference herein).

As used herein, an “antisense” nucleic acid comprises a nucleotidesequence which is complementary to a “sense” nucleic acid encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, complementary to an mRNA sequence or complementary to thecoding strand of a gene. Accordingly, an antisense nucleic acid canhydrogen bond to a sense nucleic acid.

In one embodiment, a nucleic acid molecule of the invention is an siRNAmolecule. In another embodiment, a nucleic acid molecule of theinvention is an shRNA molecule. In one embodiment, a nucleic acidmolecule of the invention mediates RNAi.

In another embodiment, a nucleic acid molecule of the invention mediatestranslational inhibition. RNA interference (RNAi) is apost-transcriptional, targeted gene-silencing technique that usesdouble-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containingthe same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287.2431-2432 (2000); Zamore, P. D., et al. Cell 101, 25-33 (2000). Tuschl,T. et al. Genes Dev. 13, 3191-3197 (1999); Cottrell T R, and Doering TL. 2003. Trends Microbiol. 11:37-43; Bushman F. 2003. MoI Therapy.7:9-10; McManus M T and Sharp P A. 2002. Nat Rev Genet. 3:737-47). Theprocess occurs when an endogenous ribonuclease cleaves the longer dsRNAinto shorter, e.g., 21- or 22-nucleotide-long RNAs, termed smallinterfering RNAs or siRNAs. The smaller RNA segments then mediate thedegradation of the target mRNA. Kits for synthesis of RNAi arecommercially available from, e.g., New England Biolabs or Ambion. In oneembodiment one or more of the chemistries described herein for use inantisense RNA can be employed in molecules that mediate RNAi.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes or nucleic acid molecules to whichthey are operatively linked and are referred to as “expression vectors”or “recombinant expression vectors” or simply “expression vectors”.Nucleic acid sequences necessary for expression in prokaryotes usuallyinclude a promoter, an operator (optional), and a ribosome binding site,often along with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals. Insome embodiments, “expression vectors” are used in order to permitpseudotyping of the viral envelope proteins.

Expression vectors are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenoviruses,adeno-associated viruses, lentiviruses), which serve equivalentfunctions.

As used herein, the term “retrovirus” is used in reference to RNAviruses that utilize reverse transcriptase during their replicationcycle. The retroviral genomic RNA is converted into double-stranded DNAby reverse transcriptase. This double-stranded DNA form of the virus iscapable of being integrated into the chromosome of the infected cell;once integrated, it is referred to as a “provirus.” The provirus servesas a template for RNA polymerase II and directs the expression of RNAmolecules which encode the structural proteins and enzymes needed toproduce new viral particles. At each end of the provirus are structurescalled “long terminal repeats” or “LTRs.” LTRs contain numerousregulatory signals, including transcriptional control elements,polyadenylation signals, and sequences needed for replication andintegration of the viral genome. LTRs may be several hundred base pairsin length.

The term “AAV vector” refers to a vector derived from anadeno-associated virus serotype, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, or AAVX7. “rAAV vector” refers to a vectorthat includes AAV nucleotide sequences as well as heterologousnucleotide sequences. rAAV vectors require only the 145 base terminalrepeats in cis to generate virus. All other viral sequences aredispensable and may be supplied in trans (Muzyczka (1992) Curr. TopicsMicrobiol. Immunol. 158:97). Typically, the rAAV vector genome will onlyretain the inverted terminal repeat (ITR) sequences so as to maximizethe size of the transgene that can be efficiently packaged by thevector. The ITRs need not be the wild-type nucleotide sequences, and maybe altered, e.g., by the insertion, deletion or substitution ofnucleotides, as long as the sequences provide for functional rescue,replication and packaging. In particular embodiments, the AAV vector isan AAV2/5 or AAV2/8 vector. Suitable AAV vectors are described in, forexample, U.S. Pat. No. 7,056,502 and Yan et al. (2002) J. Virology76(5):2043-2053, the entire contents of which are incorporated herein byreference.

As used herein, the term “lentivirus” refers to a group (or genus) ofretroviruses that give rise to slowly developing disease. Virusesincluded within this group include HIV (human immunodeficiency virus;including but not limited to HIV type 1 and HIV type 2), the etiologicagent of the human acquired immunodeficiency syndrome (AIDS);visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) insheep; the caprine arthritis-encephalitis virus, which causes immunedeficiency, arthritis, and encephalopathy in goats; equine infectiousanemia virus (EIAV), which causes autoimmune hemolytic anemia, andencephalopathy in horses; feline immunodeficiency virus (FIV), whichcauses immune deficiency in cats; bovine immune deficiency virus (BIV),which causes lymphadenopathy, lymphocytosis, and possibly centralnervous system infection in cattle; and simian immunodeficiency virus(SIV), which cause immune deficiency and encephalopathy in sub-humanprimates. Diseases caused by these viruses are characterized by a longincubation period and protracted course. Usually, the viruses latentlyinfect monocytes and macrophages, from which they spread to other cells.HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T-cells). Inone embodiment of the invention, the lentivirus is not HIV.

The term “promoter” as used herein refers to a recognition site of a DNAstrand to which the RNA polymerase binds. The promoter forms aninitiation complex with RNA polymerase to initiate and drivetranscriptional activity. The complex can be modified by activatingsequences termed “enhancers” or inhibitory sequences termed “silencers”.

The terms “transformation,” “transfection,” and “transduction” refer tointroduction of a nucleic acid, e.g., a viral vector, into a recipientcell.

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the primateis a human.

As used herein, the various forms of the term “modulate” are intended toinclude stimulation (e.g., increasing or upregulating a particularresponse or activity) and inhibition (e.g., decreasing or downregulatinga particular response or activity).

As used herein, the term “contacting” (i.e., contacting a cell with anagent) is intended to include incubating the agent and the cell togetherin vitro (e.g., adding the agent to cells in culture) or administeringthe agent to a subject such that the agent and cells of the subject arecontacted in vivo. The term “contacting” is not intended to includeexposure of cells to an agent that may occur naturally in a subject(i.e., exposure that may occur as a result of a natural physiologicalprocess).

As used herein, the term “administering” to a subject includesdispensing, delivering or applying a composition of the invention, e.g.,capable of inhibiting oxidative stress, to a subject by any suitableroute for delivery of the composition to the desired location in thesubject, including delivery by intraocular administration or intravenousadministration. Alternatively or in combination, delivery is by thetopical, parenteral or oral route, intracerebral injection,intramuscular injection, subcutaneous/intradermal injection, intravenousinjection, buccal administration, transdermal delivery andadministration by the rectal, colonic, vaginal, intranasal orrespiratory tract route.

Various additional aspects of the methods of the invention are describedin further detail in the following subsections.

Methods of the Invention

The present invention provides methods for inhibiting oxidative stressof a photoreceptor cell, which generally comprise contacting aphotoreceptor cell with an agent which increases the expression and/oractivity of an antioxidant defense protein.

The present invention also provides methods for treating or preventing aretinal disorder, e.g., a retinal disorder associated with oxidativestress of a photoreceptor cell, in a subject. The methods generallycomprise administering to the subject an agent which increases theexpression and/or activity of an antioxidant defense protein.

In another aspect, the present invention provides methods for treatingor preventing retinitis pigmentosa in a subject. Such methods generallycomprise administering to the subject an agent which increases theexpression and/or activity of an antioxidant defense protein.

The present invention further provides methods for prolonging theviability of a photoreceptor cell, e.g., a photoreceptor cellcompromised by a disorder associated with oxidative stress. The methodsgenerally comprise contacting the cell with an agent which increases theexpression and/or activity of an antioxidant defense protein.

In the methods of the invention, a cell may be contacted with or asubject administered a single antioxidant defense genes or a combinationof antioxidant defense proteins. Suitable antioxidant defense genesinclude catalase, superoxide dismutase 2 (SOD2), peroxisomeproliferator-activated receptor gamma coactivator 1-alpha (PGC1α),nuclear factor erythroid 2-like 2 (nrf2). Suitable combinations ofantioxidant defense genes for use in the methods of the inventioninclude catalase; SOD2; PGC1α; nrf2; catalase and SOD2; SOD2 and PGC1α;PGC1α and nrf2; catalase and PGC1α; catalase and nrf2; SOD2 and nrf2;catalase, SOD2, and PGC1α; SOD2, PGC1α, and nrf2; catalase, PGC1α, andnrf2; catalase, SOD2, and nrf2; or catalase, SOD2, PGC1α, and nrf2.

In one embodiment, the methods described herein can be performed invitro. For example, in on embodiment, intracellular levels of anantioxidant defense protein (e.g., catalase, SOD2, PGC1α, and/or nrf2)can be modulated in a cell in vitro and then the treated cells can beadministered or re-administered to a subject. In one embodiment, thecell is a mammalian cell, e.g., a human cell. For practicing the methodsin vitro, cells can be obtained from a subject by standard methods andincubated (e.g., cultured) in vitro with an agent which stimulatesintracellular levels of an antioxidant defense protein (e.g., catalase,SOD2, PGC1α, and/or nrf2). Methods for isolating cells are well known inthe art. The cells can be re-administered to the same subject, oranother subject which is compatible with the donor of the cells.

For administration of cells to a subject, it may be preferable to firstremove residual agents in the culture from the cells beforeadministering them to the subject. This can be done, for example, bygradient centrifugation of the cells or by washing of the tissue.Methods for the ex vivo genetic modification of cells followed byre-administration to a subject are well known in the art and describedin, for example, U.S. Pat. No. 5,399,346 the entire contents of whichare incorporated herein by reference.

In one embodiment, the invention provides methods for modulation ofintracellular levels of an antioxidant defense protein (e.g., catalase,SOD2, PGC1α, and/or nrf2) in vivo, by administering to a subject atherapeutically effective amount of an agent as described herein. Forexample, intracellular levels of catalase, SOD2, PGC1α, and/or nrf2 canbe modulated to treat or prevent a retinal disorder, such as a retinaldisorder associated with cellular oxidative stress of a photoreceptorcells.

The claimed methods of modulation are not meant to include naturallyoccurring events. For example, the term “agent” or “modulator” is notmeant to embrace endogenous mediators produced by the cells of asubject.

Application of the methods of the invention for the treatment and/orprevention of a disorder can result in curing the disorder, decreasingat least one symptom associated with the disorder, either in the longterm or short term or simply a transient beneficial effect to thesubject. Accordingly, as used herein, the terms “treat,” “treatment” and“treating” include the application or administration of agents, asdescribed herein, to a subject who is suffering from a retinal disorder,e.g., associated with oxidative stress of a photoreceptor cell, or whois susceptible to such conditions with the purpose of curing, healing,alleviating, relieving, altering, remedying, ameliorating, improving oraffecting such conditions or at least one symptom of such conditions. Asused herein, the condition is also “treated” if recurrence of thecondition is reduced, slowed, delayed or prevented.

Subjects suitable for treatment using the regimens of the presentinvention should have or are susceptible to developing retinal disordersassociated with oxidative stress. For example, subjects may begenetically predisposed to development of the disorders. Alternatively,abnormal progression of the following factors including, but not limitedto visual acuity, the rate of death of cone and/or rod cells, nightvision, peripheral vision, attenuation of the retinal vessels, and otherophthalmoscopic factors associated with retinal disorders such asretinitis pigmentosa may indicate the existence of or a predispositionto a retinal disorder.

The agents, as described herein, may be administered as necessary toachieve the desired effect and depend on a variety of factors including,but not limited to, the severity of the condition, age and history ofthe subject and the nature of the composition, for example, the identityof the genes or the affected biochemical pathway. In variousembodiments, the compositions may be administered at least two, three,four, five or six times a day. Additionally, the therapeutic orpreventative regimens may cover a period of at least about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24weeks.

The ability of an agent to up-regulate intracellular levels of catalase,SOD2, PGC1α, and/or nrf2 can be determined as described herein, e.g., bydetermining the ability of the agent to modulate cell viability (e.g.,modulation of apoptosis), cleavage of LaminA or Caspase 3; production ofreactive oxygen species; assessment for oxidation usingimmunohistochemical detection of acrolein, as well as ELISA for carbonyladducts in retinal extracts upon exposure of cells to an oxidant (e.g.,paraquat); and/or the expression and protein synthesis of photoreceptorspecific opsins.

In various embodiments, the methods of the present invention furthercomprise monitoring the effectiveness of treatment. For example, visualacuity, the rate of death of cone and/or rod cells, night vision,peripheral vision, attenuation of the retinal vessels, and otherophthalmoscopic changes associated with retinal disorders such asretinitis pigmentosa may be monitored to assess the effectiveness oftreatment. Additionally, the rate of death of cells associated with theparticular disorder that is the subject of treatment and/or prevention,may be monitored. Alternatively, the viability of such cells may bemonitored, for example, as measured by phospholipid production. Theassays described in the Examples section below may also be used tomonitor the effectiveness of treatment (e.g., electroretinography—ERG).

In one embodiment, an agent for use in the methods of the presentinvention is a nucleic acid molecule encoding an antioxidant defenseprotein, e.g., superoxide dismutase 2 (SOD2), catalase, peroxisomeproliferator-activated receptor gamma coactivator 1-alpha (PGC1α), andnuclear factor erythroid 2-like 2 (nrf2) (or combinations thereof).

In one embodiment, the nucleic acid molecule encodes superoxidedismutase 2 (SOD2). SOD2 is a member of the iron/manganese superoxidedismutase family. It encodes a mitochondrial protein that forms ahomotetramer and binds one manganese ion per subunit. This protein bindsto the superoxide byproducts of oxidative phosphorylation and convertsthem to hydrogen peroxide and diatomic oxygen. There are threealternative transcripts of SOD2, the amino acid sequences of which areknown and may be found in, for example, GenBank Accession Nos.GI:67782304, GI:67782306, and GI:67782308.

In another embodiment, the nucleic acid molecule encodes catalase.Catalase is a heme enzyme that is present in the peroxisome of nearlyall aerobic cells. Catalase converts the reactive oxygen specieshydrogen peroxide to water and oxygen and thereby mitigates the toxiceffects of hydrogen peroxide. The amino acid sequence of catalase isknown and may be found in, for example, GenBank Accession No. GI:260436906.

In another embodiment, the nucleic acid molecule encodes peroxisomeproliferator-activated receptor gamma coactivator 1-alpha (PGC1α). PGC1αis a transcriptional co-activator that regulates the genes involved inenergy metabolism. PGC1α interacts with PPARgamma, which permits theinteraction of PGC1α with multiple transcription factors. PGC1α caninteract with, and regulate the activities of, cAMP response elementbinding protein (CREB) and nuclear respiratory factors (NRFs) [Here iscorrect to keep nuclear respiratory factors]. It provides a direct linkbetween external physiological stimuli and the regulation ofmitochondrial biogenesis. The amino acid sequence of PGC1α is known andmay be found in, for example, GenBank Accession No. GI:116284374.

In another embodiment, the nucleic acid molecule encodes Nuclear factor(erythroid-derived 2)-like 2 (nrf2), a transcription factor which is amember of a small family of basic leucine zipper (bZIP) proteins. Theencoded transcription factor regulates genes which contain antioxidantresponse elements (ARE) in their promoters. There are three alternativetranscripts of nrf2, the amino acid sequences of which are known and maybe found in, for example, GenBank Accession Nos. GI:372620347,GI:372620348, and GI:372620346.

In one embodiment, the viability or survival of photoreceptor cells,such as cones cells, is short term viability, e.g., about 1 week, about2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks,about 7 weeks, about 8 weeks, about 3 years, about 4 years, about 5years, about 10 years, about 15, years, about 20 years, about 25 years,about 30 years, about 40 years, about 50 years, about 60 years, about 70years, and about 80 years.

The methods of the invention described above, thus, may be used to treator prevent oxidative stress of photoreceptor cells and associateddisorders. In one embodiment, the disorder is a retinal disorderincluding, but not limited to, retinitis pigmentosa, age related maculardegeneration, cone rod dystrophy, and rod cone dystrophy. In otherembodiments, the retinal disorder is not associated with blood vesselleakage and/or growth. In certain embodiments, the retinal disorder isnot associated with diabetes. In another embodiment, the retinaldisorder is not diabetic retinopathy. In further embodiments, theretinal disorder is not NARP (neuropathy, ataxia and retinitispigmentosa). In one embodiment, the disorder is a retinal disorderassociated with decreased viability of cone and/or rod cells. In yetanother embodiment, the disorder is a genetic disorder.

In one embodiment, the invention is directed to a method of treating orpreventing a retinal disorder, such as a retinal disorder associatedwith oxidative stress of a photoreceptor cell, for example, retinitispigmentosa, in a subject by selecting a subject who is susceptible tothe development of the disorder and administering to the subject aneffective amount of the nucleic acid molecules, vectors and/orcompositions of the present invention, thereby treating or preventingthe disorder in the subject.

The overall strategy to save photoreceptor cells from degeneration byexposure to reactive oxygen species as a result of oxidative stress isto supply them with the genes that will allow them to reduce oreliminate the hyperoxic environment by, e.g., inhibiting and/or removingreactive oxygen species by increasing the expression and/or activity ofantioxidant defense protein(s).

In general, the nucleic acid molecules and/or the vectors of theinvention are provided in a therapeutically effective amount to elicitthe desired effect, e.g., inhibit oxidative stress in a photoreceptorcell. The quantity of the nucleic acid molecule, and/or vector to beadministered, both according to number of treatments and amount, willalso depend on factors such as the clinical status, age, and weight ofthe subject to be treated, and the severity of the disorder. Preciseamounts of active ingredient required to be administered depend on thejudgment of the gene therapist and will be particular to each individualpatient. For example, a viral vector comprising the nucleic acidmolecules of the invention is administered in titers ranging from about1×10⁵ to about 1×10⁹ colony forming units (cfu) per ml, although rangesmay vary. Preferred titers will range from about 1×10⁶ to about 1×10⁸cfu/ml.

A therapeutically effective amount of the nucleic acid molecules and/orthe vectors of the invention (i.e., an effective dosage) may range fromabout 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kgbody weight, more preferably about 0.1 to 20 mg/kg body weight, and evenmore preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciatethat certain factors may influence the dosage required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of the nucleic acidmolecules and/or the vectors of the invention can include a singletreatment or, preferably, can include a series of treatments. It willalso be appreciated that the effective dosage used for treatment mayincrease or decrease over the course of a particular treatment. Changesin dosage may result from the results of diagnostic assays as describedherein. The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

The term “prophylactic” or “therapeutic” treatment refers toadministration to the subject of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if administeredafter manifestation of the unwanted condition, the treatment istherapeutic (i.e., it is intended to diminish, ameliorate or maintainthe existing unwanted condition or side effects therefrom).

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of a nucleic acid molecule and/or the vectors of theinvention that, when administered to a patient for treating aneurodegenerative disease, is sufficient to effect treatment of thedisease (e.g., by diminishing, ameliorating or maintaining the existingdisease or one or more symptoms of disease). The “therapeuticallyeffective amount” may vary depending on the nucleic acid molecule,peptide, and/or the vector, how the nucleic acid molecule and/or thevectors is administered, the disease and its severity and the history,age, weight, family history, genetic makeup, stage of pathologicalprocesses mediated by the neurodegenerative disease expression, thetypes of preceding or concomitant treatments, if any, and otherindividual characteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of a nucleic acid molecule and/or the vector that,when administered to a subject who does not yet experience or displaysymptoms of e.g., a retinal disorder, but who may be predisposed to thedisease, is sufficient to prevent or ameliorate the disease or one ormore symptoms of the disease. Ameliorating the disease includes slowingthe course of the disease or reducing the severity of later-developingdisease. The “prophylactically effective amount” may vary depending onthe nucleic acid molecule and/or the vector, how the nucleic acidmolecule and/or the vector is administered, the degree of risk ofdisease, and the history, age, weight, family history, genetic makeup,the types of preceding or concomitant treatments, if any, and otherindividual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of a nucleic acid molecule and/or thevector that produces some desired local or systemic effect at areasonable benefit/risk ratio applicable to any treatment. A nucleicacid molecule and/or the vector employed in the methods of the presentinvention may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms, diminishing the extent ofinfection, stabilized (i.e., not worsening) state of infection,amelioration or palliation of the infectious state, whether detectableor undetectable. “Treatment” can also mean prolonging survival ascompared to expected survival in the absence of treatment.

In certain embodiments of the invention, an agent, e.g., an isolatednucleic acid molecule and/or vector of the invention, is administered incombination with an additional therapeutic agent or treatment. Thecompositions and an additional therapeutic agent can be administered incombination in the same composition or the additional therapeutic agentcan be administered as part of a separate composition or by anothermethod described herein.

Examples of additional therapeutic agents suitable for use in themethods of the invention include those agents known to treat retinaldisorders, such as retinitis pigmentosa and age-related maculardegeneration and include, for example, fat soluble vitamins (e.g.,vitamin A, vitamin E, and ascorbic acid), calcium channel blockers(e.g., diltiazem) carbonic anhydrase inhibitors (e.g., acetazolamide andmethazolamide), anti-angiogenics (e.g., antiVEGF antibodies), growthfactors (e.g., rod-derived cone viability factor (RdCVF), BDNF, CNTF,bFGF, and PEDF), antioxidants, other gene therapy agents (e.g.,optogenetic gene threrapy, e.g., channelrhodopsin, melanopsin, andhalorhodopsin), and compounds that drive photoreceptor regeneration by,e.g., reprogramming Müller cells into photoreceptor progenitors (e.g.,alpha-aminoadipate). Exemplary treatments for use in combination withthe treatment methods of the present invention include, for example,retinal and/or retinal pigmented epithelium transplantation, stem celltherapies, retinal prostheses, laser photocoagulation, photodynamictherapy, low vision aid implantation, submacular surgery, and retinaltranslocation.

Agents for Use in the Methods of the Invention

Stimulatory Agents

The methods of the invention may use stimulatory agents which increasethe expression and/or activity of an antioxidant defense protein in acell. Examples of such stimulatory agents include proteins, nucleic acidmolecules, e.g., expression vectors comprising nucleic acid molecules,and chemical agents that stimulate expression and/or activity of aprotein which increases the expression and/or activity of an antioxidantdefense protein in a cell.

A preferred stimulatory agent is a nucleic acid molecule encoding aprotein of interest. For example, a cDNA (full length or partial cDNAsequence) is cloned into a recombinant expression vector and the vectoris transfected into cells using standard molecular biology techniques.The cDNA can be obtained, for example, by amplification using thepolymerase chain reaction (PCR) or by screening an appropriate cDNAlibrary.

Following isolation or amplification of a cDNA, the DNA fragment isintroduced into a suitable expression vector. For example, nucleic acidmolecules encoding a protein of interest in the form suitable forexpression of the protein in a host cell, can be prepared usingnucleotide sequences based on the nucleic acid sequence of a nucleicacid molecule encoding the protein of interest.

In one embodiment, a stimulatory agent can be present in an inducibleconstruct. In another embodiment, a stimulatory agent can be present ina construct which leads to constitutive expression.

In one embodiment, the nucleic acid molecules of the invention may bedelivered to cells, e.g., photoreceptor cells, or to subjects, in avector, e.g., a recombinant expression vector. In another embodiment,the nucleic acid molecules of the invention may be delivered to cells,e.g., photoreceptor cells, or to subjects, in the absence of a vector.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably. However, the invention is intendedto include such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells, those which areconstitutively active, those which are inducible, and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or portions thereof, including fusion proteins orportions thereof, encoded by nucleic acids as described herein.

In one embodiment, a nucleic acid molecule encoding an antioxidantdefense protein is expressed in mammalian cells using a mammalianexpression vector. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J 6:187-195). When used in mammalian cells, the expression vector'scontrol functions are often provided by viral regulatory elements.

In certain embodiments, the nucleic acid molecules of the invention arecontained within a viral vector and may be delivered to cells, e.g.,photoreceptor cells, or to subjects. Preferably a viral vector is onewhose use for gene therapy is well known in the art. Techniques for theformation of vectors or virions are generally described in “WorkingToward Human Gene Therapy,” Chapter 28 in Recombinant DNA, 2nd Ed.,Watson, J. D. et al., eds., New York: Scientific American Books, pp.567-581 (1992). An overview of suitable viral vectors or virions isprovided in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. 1):31-32(1997), as well as Nakanishi, M., Crit. Rev. Therapeu. Drug CarrierSystems 12:263-310 (1995); Robbins, P. D., et al., Trends Biotechnol.16:35-40 (1998); Zhang, J., et al., Cancer Metastasis Rev.15:385-401(1996); and Kramm, C. M., et al., Brain Pathology 5:345-381(1995). Such vectors may be derived from viruses that contain RNA (Vile,R. G., et al., Br. Med Bull. 51:12-30 (1995)) or DNA (Ali M., et al.,Gene Ther. 1:367-384 (1994)).

Examples of viral vector systems utilized in the gene therapy art and,thus, suitable for use in the present invention, include the following:retroviruses (Vile, R. G., supra; U.S. Pat. Nos. 5,741,486 and5,763,242); adenoviruses (Brody, S. L., et al., Ann. N.Y. Acad. Sci.716: 90-101 (1994); Heise, C. et al., Nat. Med. 3:639-645 (1997));adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624-634(1997); Feng, M., et al., Nat. Biotechnol. 15:866-870 (1997));adeno-associated viruses (Flotte, T. R. and Carter, B. J., Gene Ther.2:357-362 (1995); U.S. Pat. No. 5,756,283); herpes simplex virus I or II(Latchman, D. S., Mol. Biotechnol. 2:179-195 (1994); U.S. Pat. No.5,763,217; Chase, M., et al., Nature Biotechnol. 16:444-448 (1998));parvovirus (Shaughnessy, E., et al., Semin Oncol. 23:159-171 (1996));reticuloendotheliosis virus (Donburg, R., Gene Therap. 2:301-310(1995)). Extrachromosomal replicating vectors may also be used in thegene therapy methods of the present invention. Such vectors aredescribed in, for example, Calos, M. P. (1996) Trends Genet. 12:463-466,the entire contents of which are incorporated herein by reference. Otherviruses that can be used as vectors for gene delivery includepoliovirus, papillomavirus, vaccinia virus, lentivirus, as well ashybrid or chimeric vectors incorporating favorable aspects of two ormore viruses (Nakanishi, M. (1995) Crit. Rev. Therapeu. Drug CarrierSystems 12:263-310; Zhang, J., et al. (1996) Cancer Metastasis Rev.15:385-401; Jacoby, D. R., et al. (1997) Gene Therapy 4:1281-1283).

As used herein, the term “retrovirus” is used in reference to RNAviruses that utilize reverse transcriptase during their replicationcycle. The retroviral genomic RNA is converted into double-stranded DNAby reverse transcriptase. This double-stranded DNA form of the virus iscapable of being integrated into the chromosome of the infected cell;once integrated, it is referred to as a “provirus.” The provirus servesas a template for RNA polymerase II and directs the expression of RNAmolecules which encode the structural proteins and enzymes needed toproduce new viral particles. At each end of the provirus are structurescalled “long terminal repeats” or “LTRs.” LTRs contain numerousregulatory signals, including transcriptional control elements,polyadenylation signals, and sequences needed for replication andintegration of the viral genome. LTRs may be several hundred base pairsin length.

The term “AAV vector” refers to a vector derived from anadeno-associated virus serotype, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, or AAVX7. “rAAV vector” refers to a vectorthat includes AAV nucleotide sequences as well as heterologousnucleotide sequences. rAAV vectors require only the 145 base terminalrepeats in cis to generate virus. All other viral sequences aredispensable and may be supplied in trans (Muzyczka (1992) Curr. TopicsMicrobiol. Immunol. 158:97). Typically, the rAAV vector genome will onlyretain the inverted terminal repeat (ITR) sequences so as to maximizethe size of the transgene that can be efficiently packaged by thevector. The ITRs need not be the wild-type nucleotide sequences, and maybe altered, e.g., by the insertion, deletion or substitution ofnucleotides, as long as the sequences provide for functional rescue,replication and packaging. In particular embodiments, the AAV vector isan AAV2/5 or AAV2/8 vector. Suitable AAV vectors are described in, forexample, U.S. Pat. No. 7,056,502 and Yan et al. (2002) J. Virology76(5):2043-2053, the entire contents of which are incorporated herein byreference.

As used herein, the term “lentivirus” refers to a group (or genus) ofretroviruses that give rise to slowly developing disease. Virusesincluded within this group include HIV (human immunodeficiency virus;including but not limited to HIV type 1 and HIV type 2), the etiologicagent of the human acquired immunodeficiency syndrome (AIDS);visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) insheep; the caprine arthritis-encephalitis virus, which causes immunedeficiency, arthritis, and encephalopathy in goats; equine infectiousanemia virus (EIAV), which causes autoimmune hemolytic anemia, andencephalopathy in horses; feline immunodeficiency virus (FIV), whichcauses immune deficiency in cats; bovine immune deficiency virus (BIV),which causes lymphadenopathy, lymphocytosis, and possibly centralnervous system infection in cattle; and simian immunodeficiency virus(SIV), which cause immune deficiency and encephalopathy in sub-humanprimates. Diseases caused by these viruses are characterized by a longincubation period and protracted course. Usually, the viruses latentlyinfect monocytes and macrophages, from which they spread to other cells.HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T-cells). Inone embodiment of the invention, the lentivirus is not HIV.

As used herein, the term “adenovirus” (“Ad”) refers to a group ofdouble-stranded DNA viruses with a linear genome of about 36 kb. See,e.g., Berkner et al., Curr. Top. Microbiol. Immunol., 158: 39-61 (1992).In some embodiments, the adenovirus-based vector is an Ad-2 or Ad-5based vector. See, e.g., Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-123, 1992; Ali et al., 1994 Gene Therapy 1: 367-384; U.S. Pat. Nos.4,797,368, and 5,399,346. Suitable adenovirus vectors derived from theadenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art.Recombinant adenoviruses are advantageous in that they do not requiredividing cells to be effective gene delivery vehicles and can be used toinfect a wide variety of cell types. Additionally, introduced adenovirusDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenovirus genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Haj-Ahmand et al. J. Virol. 57, 267-273[1986]).

In one embodiment, an adenovirus is a replication defective adenovirus.Most replication-defective adenoviral vectors currently in use have allor parts of the viral E1 and E3 genes deleted but retain as much as 80%of the adenovirus genetic material. Adenovirus vectors deleted for allviral coding regions are also described by Kochanek et al. andChamberlain et al. (U.S. Pat. Nos. 5,985,846 and 6,083,750). Suchviruses are unable to replicate as viruses in the absence of viralproducts provided by a second virus, referred to as a “helper” virus.

In one embodiment, an adenoviral vector is a “gutless” vector. Suchvectors contain a minimal amount of adenovirus DNA and are incapable ofexpressing any adenovirus antigens (hence the term “gutless”). Thegutless replication defective Ad vectors provide the significantadvantage of accommodating large inserts of foreign DNA while completelyeliminating the problem of expressing adenoviral genes that result in animmunological response to viral proteins when a gutless replicationdefective Ad vector is used in gene therapy. Methods for producinggutless replication defective Ad vectors have been described, forexample, in U.S. Pat. No. 5,981,225 to Kochanek et al., and U.S. Pat.Nos. 6,063,622 and 6,451,596 to Chamberlain et al; Parks et al., PNAS93:13565 (1996) and Lieber et al., J. Virol. 70:8944-8960 (1996).

In another embodiment, an adenoviral vector is a “conditionallyreplicative adenovirus” (“CRAds”). CRAds are genetically modified topreferentially replicate in specific cells by either (i) replacing viralpromoters with tissue specific promoters or (ii) deletion of viral genesimportant for replication that are compensated for by the target cellsonly. The skilled artisan would be able to identify epithelial cellspecific promoters.

Other art known adenoviral vectors may be used in the methods of theinvention. Examples include Ad vectors with recombinant fiber proteinsfor modified tropism (as described in, e.g., van Beusechem et al., 2000Gene Ther. 7: 1940-1946), protease pre-treated viral vectors (asdescribed in, e.g., Kuriyama et al., 2000 Hum. Gene Ther. 11:2219-2230), E2a temperature sensitive mutant Ad vectors (as describedin, e.g., Engelhardt et al., 1994 Hum. Gene Ther. 5: 1217-1229), and“gutless” Ad vectors (as described in, e.g., Armentano et al., 1997 J.Virol. 71: 2408-2416; Chen et al., 1997 Proc. Nat. Acad. Sci. USA 94:1645-1650; Schieder et al., 1998 Nature Genetics 18: 180-183).

In a particular embodiment, the viral vector for use in the methods ofthe present invention is an AAV vector. In particular embodiments, theviral vector is an AAV2/5 or AAV2/8 vector. Such vectors are describedin, for example, U.S. Pat. No. 7,056,502, the entire contents of whichare incorporated herein by reference. In another embodiment, adenoviralvectors suitable for use in the present invention may include those thatare capable of transducing all retinal cell types upon intravitrealadministration, such as an AAV2 variant having a V708I mutation.Additional suitable adenoviral vectors are those that do not generate ahumoral immune response against the viral capsid upon administration(see, e.g., Dalkara et al, 2013 Sci Transl Med. 5, 189ra76), and thosethat facilitate nuclear transport of the AAV vector by, e.g., reducingubiquitination and proteasome-mediated degradation of the vector, suchas vectors having mutations that prevent phosphorylation of tyrosineresidues in AAV capsid proteins (as described in e.g., Pang et al, 2010The American Society of Gene & Cell Therapy. 19, 2: 234-242).

The vector will include one or more promoters or enhancers, theselection of which will be known to those skilled in the art. Forexample, commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus and simian virus 40. Suitable promoters include, but arenot limited to, the retroviral long terminal repeat (LTR), the SV40promoter, the human cytomegalovirus (CMV) promoter, and other viral andeukaryotic cellular promoters known to the skilled artisan. For othersuitable expression systems for both prokaryotic and eukaryotic cellssee chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In another embodiment, the viral vector is capable of directingexpression of the nucleic acid preferentially in a particular cell type(e.g., tissue-specific regulatory elements are used to express thenucleic acid). Tissue-specific regulatory elements are known in the art.In one embodiment, a tissue-specific promoter for use in the vectors andmethods of the invention is a retinal cell-specific promoter. In oneembodiment, a retinal cell-specific promoter is a rod-, cone-, andbipolar cell-specific promoter. In one embodiment, a retinalcell-specific promoter is a rod- and cone-specific promoter. In oneembodiment, a retinal cell-specific promoter is a rod-specific promoter.In one embodiment, a retinal cell-specific promoter is a cone-specificpromoter.

Suitable retinal cell-specific promoters are known in the art andinclude, e.g., rhodopsin regulatory sequences, Nrl, Crx, Rax, and thelike (Matsuda and Cepko (2007) Proc. Natl. Acad. Sci. U.S.A. 104:1027),opsin promoters, e.g., cone opsin, interphotoreceptor retinoid bindingprotein promoters (IRBP156), rhodopsin kinase (RK) promoters, neuralleucine zipper (NRLL) promoters, (see, e.g., Semple-Rowland, et al.(2010) Molec Vision 16:916), or combinations thereof. Additionalsuitable promoters may include cone arrestin, Cabp5, Cralbp, Ndrg4,clusterin, Hesl, vimentin promoters, cluster differentiation (CD44)promoters, and glial fibrillary acid protein (GFAP) promoters.

Guidance in the construction of gene therapy vectors and theintroduction thereof into affected animals for therapeutic purposes maybe obtained in the above-referenced publications, as well as in U.S.Pat. Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,529,774,5,601,818, and PCT Publication No. WO 95/06486, the entire contents ofwhich are incorporated herein by reference.

Generally, methods are known in the art for transfection andtransformation of the cells of interest. For example, a virus can beplaced in contact with the neuronal cell of interest or alternatively,can be injected into a subject suffering from a neurodegenerativedisorder.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection (e.g., usingcommercially available reagents such as, for example, LIPOFECTIN®(Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE® (Invitrogen),FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™(Polyplus-transfection Inc., New York, N.Y.), EFFECTENE® (Qiagen,Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), orelectroporation (e.g., in vivo electroporation). Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring harborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

As discussed above, the nucleic acid molecules of the invention can beinserted into vectors and used as gene therapy vectors. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (see, e.g., U.S. Pat. No. 5,328,470),stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. U.S.A. 91:3054), or by in vivo electroporation (see, e.g., Matsudaand Cepko (2007) Proc. Natl. Acad. Sci. U.S.A. 104:1027). Localadministration of nucleic acids and/or gene therapy vectors describedherein can be by any suitable method in the art including, for example,injection (e.g., intravitreal or subretinal or subvitreal injection),gene gun, by topical application of the nucleic acid in a gel, oil, orcream, by electroporation, using lipid-based transfection reagents,transcleral delivery, by implantation of scleral plugs or a drugdelivery device, or by any other suitable transfection method.

In one embodiment, a packaging cell line is transduced with a retroviralvector carrying the desired nucleic acid molecule to form a producercell line. The packaging cells may be transduced by any means known inthe art, including, e.g., electroporation, CaPO₄ precipitation, or theuse of liposomes. Examples of packaging cells that may be transfectedinclude, but are not limited to, BOSC23, Bing, PE501, PA317, .PSI.-2,.PSI.-AM, PA12, T19-14X, VT-19-17-H2, .PSI.-CRE, .PSI.-CRIP, GP+E86,GP+envAm12, and DAN cell lines. Guidance on retroviral producingpackaging cells and how to construct them can be found in Short et al.,J. Neurosci. Res. 27:427-433 (1990); Miller, A. D., Human Gene Ther.1:5-14 (1990); Danos, O, “Construction of Retroviral Packaging CellLines,” in Methods in Molecular Biology (M. Collins, ed.), Vol. 8, TheHumana Press Inc., Clifton, N.J., 17-26 (1991); Murdoch, B., et al.,Gene Therapy 4:744-749 (1997); and U.S. Pat. Nos. 5,529,774 and5,591,624, the entire contents of which are incorporated herein byreference.

Retroviral vectors have may also be packaged with a vesicular stomatitisvirus (VSV) envelope glycoprotein G (“pseudotyping”). These vectors aremore stable and can be concentrated to 10⁹ cfu/ml, allowing them to beinjected directly (Burns, J. C. et al. (1993) Proc. Natl. Acad. Sci. USA90:8033-8037).

The producer cells can then be grafted near or into the desiredlocation, for example, intraocularly. Direct injection of high titerretroviral producer cells (Murdoch, B., et al., Gene Ther. 4:744-749(1997); Onodera, M., et al., Hum Gene Ther. 8:1189-1194 (1997)) allowfor efficient in situ infection with the retroviral sequences (Rainov,N. G., et al., Cancer Gene Ther. 3:99-106 (1996); Ram, Z., et al.,Cancer Res. 53:83-88 (1993)). Producer cells injected intraocularly donot generally migrate from the site of injection. Moreover, althoughthey may be rejected by the host, this does not occur for 5-10 days, bywhich time retroviral infection of nearby cells will have occurred (Ram,Z., et al., J. Neurosurg. 79:400-407 (1993)). In general, vectorproducer cell (VPC) dosages range from about 2.5×10⁸, about 1×10⁸, about1.5×10⁸, about 2×10⁸, about 2.5×10⁸, about 3×10⁸, about 3.5×10⁸, about4×10⁸, about 4.5×10⁸, about 5×10⁸, about 5.5×10⁸, about 6×10⁸, about6.5×10⁸, about 7×10⁸, about 7.5×10⁸, about 8×10⁸, about 8.5×10⁸, about9×10⁸, about 9.5×10⁸, and about 1×10⁹ VPCs. The exact amount of producercells will ultimately be determined by the skilled artisan based onnumerous factors, including, but not limited to, the availableinjectable volume, clinical status of the patient, and the severity ofthe disorder.

The exact amount of producer cells will ultimately be determined by theskilled artisan based on numerous factors, including, but not limitedto, the available injectable volume, clinical status of the patient, andthe severity of the disorder.

Preferably, the viral genomes of the viral vectors used in the inventionshould be modified to remove or limit their ability to replicate,however, replication conditional viruses will also be useful in thepresent invention, as will replicating vectors that are capable oftargeting certain cells. (See, e.g., Zhang, J. et al. (1996) CancerMetastasis Rev. 15:385-401).

The nucleic acid molecules can also be delivered using non-viral methodsfor gene transfer, preferably those whose use in gene therapy is knownin the art (Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems12:263-310 (1995); Abdallah, B., et al., Biol Cell 85:1-7 (1995); Zhang,J., et al., Cancer Metastasis Rev. 15:385-401 (1996); Philips, S. C.,Biologicals 23:13-16 (1995); Lee, R. J. and Huang, L., Crit. Rev. Ther.Drug Carrier Syst. 14:173-206 (1997)). Examples of such non-viralvectors for gene delivery include prokaryotic vectors, cationicliposomes, DNA-protein complexes, non-viral T7 autogene vectors (Chen,X., et al., Hum. Gene Ther. 9:729-736 (1998)), fusogenic liposomes,direct injection of nucleic acid (“naked DNA”), particle orreceptor-mediated gene transfer, hybrid vectors such as DNA-adenovirusconjugates or other molecular conjugates involving a non-viral and viralcomponent, starburstpolyamidoamine dendrimers (Kukowska-Latallo, J. F.,et al., Proc Natl Acad Sci USA 93:4897-4902 (1996); Tang, M. X., et al.,Bioconjug. Chem. 7:703-714 (1996)), cationic peptides (Wyman, T. B., etal., Biochemistry 36:3008-3017 (1997)), and mammalian artificialchromosomes (Ascenzioni, F., et al., Cancer Lett. 118:135-142 (1997)).

Any suitable virus usable for nucleic acid delivery may be used,including, but not limited to, adenovirus, adeno-associated virus,retroviruses and the like. For example, the LIA retrovirus may be usedto deliver nucleic acids (Cepko et al. (1998) Curr. Top. Dev. Biol.36:51; Dyer and Cepko (2001) J. Neurosci. 21:4259).

In one embodiment, a single viral vector is used to carry multiplenucleic acid molecules. In another embodiment, two viral vectors areused each carrying one or more genes of interest. If two viral vectorsare used, they can be derived from the same or a different type ofvirus, and can be administered simultaneously or sequentially (i.e.,without regard for a specific order).

Gene delivery can be enhanced by including an internal ribosome entrysite (IRES) sequence to achieve coordinate expression of multiple geneson a bicistronic message. IRESs are sequences containing 500-600 bp thatare typical of the 5′ nontransduced regions of picornaviruses, includingthe polio- and encephalomyocarditis viruses (EMCV). See, e.g., Ghattas,I. R., et al., Molecular and Cellular Biology 11:5848-5859 (1991);Morgan, R. A., et al., Nucleic Acids Research 20:1293-1299 (1992). Thisapproach has been used for efficient retroviral coexpression of the twosubunits of interleukin-12 (Tahara, H., et al., J. Immunol.154:6466-6474 (1995)). Similarly, a viral sequence, the picornavirus 2Asequence, can be used to create mRNAs encoding more than one protein.The viral 2A peptide is 16-20 amino acids and can be employed as acleavage peptide located between two proteins of interest, where itpromotes their cleavage into two separate proteins (Furler et al. GeneTher. 8:864-873 (2001). Another alternative is for the vector to containmultiple genes under the control of distinct promoters.

Generally, methods are known in the art for viral infection of the cellsof interest. The virus can be placed in contact with the neuronal cellof interest or alternatively, can be injected into a subject sufferingfrom a disorder associated with photoreceptor cell oxidative stress.

In one aspect of the invention, the therapeutic nucleic acid molecule orthe vector containing the same will be in the form of a pharmaceuticalcomposition containing a pharmaceutically acceptable carrier. As usedherein “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, the carrier is suitablefor intraocular, parenteral, intravenous, intraperitoneal, topical, orintramuscular administration. In another embodiment, the carrier issuitable for oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the gene therapy vector, use thereofin the pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In a particular embodiment, the pharmaceutical compositions of thepresent invention would be administered in the form of injectablecompositions. The vector can be prepared as an injectable, either asliquid solutions or suspensions. The preparation may also be emulsified.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH-buffering agents, adjuvants orimmunopotentiators.

In a particular embodiment, the nucleic acid molecules and/or vectorsare incorporated in a composition suitable for intraocularadministration. For example, the compositions may be designed forintravitreal, subconjuctival, sub-tenon, periocular, retrobulbar,suprachoroidal, and/or intrascleral administration, for example, byinjection, to effectively treat the retinal disorder. Additionally, asutured or refillable dome can be placed over the administration site toprevent or to reduce “wash out”, leaching and/or diffusion of the activeagent in a non-preferred direction.

Relatively high viscosity compositions, as described herein, may be usedto provide effective, and preferably substantially long-lasting deliveryof the nucleic acid molecules and/or vectors, for example, by injectionto the posterior segment of the eye. A viscosity inducing agent canserve to maintain the nucleic acid molecules and/or vectors in adesirable suspension form, thereby preventing deposition of thecomposition in the bottom surface of the eye. Such compositions can beprepared as described in U.S. Pat. No. 5,292,724, the entire contents ofwhich are hereby incorporated herein by reference.

In general, the nucleic acid molecule is provided in a therapeuticallyeffective amount to elicit the desired effect, e.g., increase theexpression and/or activity of an antioxidant defense protein (e.g.,catalase, SOD2, PGC1α, and/or nrf2). The quantity of the vector to beadministered, both according to number of treatments and amount, willalso depend on factors such as the clinical status, age, and weight ofthe subject to be treated, and the severity of the disorder. Preciseamounts of active ingredient required to be administered depend on thejudgment of the gene therapist and will be particular to each individualpatient. Generally, the viral vector is administered in titers rangingfrom about 1×10⁵, about 1.5×10⁵, about 2×10⁵, about 2.5×10⁵, about3×10⁵, about 3.5×10⁵, about 4×10⁵, about 4.5×10⁵, about 5×10⁵, about5.5×10⁵, about 6×10⁵, about 6.5×10⁵, about 7×10⁵, about 7.5×10⁵, about8×10⁵, about 8.5×10⁵, about 9×10⁵, about 9.5×10⁵, about 1×10⁶, about1.5×10⁶, about 2×10⁶, about 2.5×10⁶ about 3×10⁶ about 3.5×10⁶, about4×10⁶, about 4.5×10⁶, about 5×10⁶, about 5.5×10⁶ about 6×10⁶ about6.5×10⁶, about 7×10⁶, about 7.5×10⁶, about 8×10⁶, about 8.5×10, about9×10⁶, about 9.5×10⁶, about 1×10⁷, about 1.5×10⁷, about 2×10⁷, about2.5×10⁷ about 3×10⁷ about 3.5×10⁷, about 4×10⁷, about 4.5×10⁷, about5×10⁷, about 5.5×10⁷, about 6×10⁷, about 6.5×10⁷, about 7×10⁷, about7.5×10⁷, about 8×10⁷, about 8.5×10⁷, about 9×10⁷, about 9.5×10⁷, about1×10⁸, about 1.5×10⁸, about 2×10⁸, about 2.5×10⁸ about 3×10⁸ about3.5×10⁸, about 4×10⁸, about 4.5×10⁸, about 5×10⁸, about 5.5×10⁸, about6×10⁸, about 6.5×10⁸, about 7×10⁸, about 7.5×10⁸, about 8×10⁸, about8.5×10⁸, about 9×10⁸, about 9.5×10⁸, and about 1×10⁹ colony formingunits (cfu) per ml, although ranges may vary. Preferred titers willrange from about 1×10⁶ to about 1×10⁸ cfu/ml.

Other examples of stimulatory agents for increasing the expressionand/or activity of an antioxidant defense protein (catalase, SOD2,PGC1α, and/or nrf2) in a cell is a small molecule compound, an antibody,or other protein as described below.

Inhibitory Agents

The methods of the invention may also use agents which inhibit anegative regulator that counteracts the increase in expression and/oractivity of an antioxidant defense protein (catalase, SOD2, PGC1α,and/or nrf2). Such agents can be, for example, intracellular bindingmolecules that act to specifically inhibit the expression, processing,post-translational modification, or activity of a negative regulatorthat counteracts the increase in expression and/or activity of anantioxidant defense protein (catalase, SOD2, PGC1α, and/or nrf2). Asused herein, the term “intracellular binding molecule” is intended toinclude molecules that act intracellularly to, for example, inhibit theprocessing expression or activity of a protein by binding to the proteinor to a nucleic acid (e.g. an mRNA molecule) that encodes the protein.

Examples of intracellular binding molecules, described in further detailbelow, include antisense nucleic acids, intracellular antibodies,peptidic compounds, and chemical agents that specifically inhibit theactivity of a negative regulator that counteracts the increase inexpression and/or activity of an antioxidant defense protein (catalase,SOD2, PGC1α, and/or nrf2).

In one embodiment, such an agent is an antisense nucleic acid moleculethat is complementary to a gene encoding a negative regulator thatcounteracts the increase in expression and/or activity of an antioxidantdefense protein (catalase, SOD2, PGC1α, and/or nrf2), or to a portion ofthe gene, or a recombinant expression vector encoding the antisensenucleic acid molecule. The use of antisense nucleic acids todownregulate the expression of a particular protein in a cell is wellknown in the art (see e.g. Weintraub, H. et al., Antisense RNA as amolecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986; Askari, F. K. and McDonnell, W. M. (1996) N. Eng J. Med.334:316-318; Bennett, M. R. and Schwartz, S. M. (1995) Circulation92:1981-1993; Mercola, D. and Cohen, J. S. (1995) Cancer Gene Ther.:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Wagner, R. W.(1994) Nature 372:333-335; each of which is incorporated herein byreference). An antisense nucleic acid molecule comprises a nucleotidesequence that is complementary to the coding strand of another nucleicacid molecule (e. g., an mRNA sequence) and accordingly is capable ofhydrogen bonding to the coding strand of the other nucleic acidmolecule.

Antisense sequences complementary to a sequence of an mRNA can becomplementary to a sequence found in the coding region of the mRNA, the5′ or 3′ untranslated region of the mRNA or a region bridging the codingregion and an untranslated region (e.g. at the junction of the 5′untranslated region and the coding region). Furthermore, an antisensenucleic acid can be complementary in sequence to a regulatory region ofthe gene encoding the mRNA, for instance a transcription initiationsequence or regulatory element. Preferably, an antisense nucleic acid isdesigned so as to be complementary to a region preceding or spanning theinitiation codon on the coding strand or in the 3′ untranslated regionof an mRNA.

Antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The anti sense nucleic acidmolecule can be complementary to the entire coding region of an mRNA,but more preferably is antisense to only a portion of the coding ornoncoding region of an mRNA.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleicacid of the invention can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g. an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. To inhibit expression in cells, one or moreantisense oligonucleotides can be used.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which all or a portion of a cDNA hasbeen subcloned in an antisense orientation (i.e., nucleic acidtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest). Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the expression of the antisense RNA moleculein a cell of interest, for instance promoters and/or enhancers or otherregulatory sequences can be chosen which direct constitutive, tissuespecific or inducible expression of antisense RNA. The antisenseexpression vector is prepared according to standard recombinant DNAmethods for constructing recombinant expression vectors, except that thecDNA (or portion thereof) is cloned into the vector in the antisenseorientation. The antisense expression vector can be in the form of, forexample, a recombinant plasmid, phagemid or attenuated virus. Theantisense expression vector can be introduced into cells using astandard transfection technique.

Antisense nucleic acid molecules are typically administered to a subjector generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a protein to thereby inhibit expressionof the protein, e.g. by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarily to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of an antisense nucleic acid molecule of the inventionincludes direct injection at a tissue site. Alternatively, an antisensenucleic acid molecule can be modified to target selected cells and thenadministered systemically. For example, for systemic administration, anantisense molecule can be modified such that it specifically binds to areceptor or an antigen expressed on a selected cell surface, e.g. bylinking the antisense nucleic acid molecule to a peptide or an antibodywhich binds to a cell surface receptor or antigen. The antisense nucleicacid molecule can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, an antisense nucleic acid molecule that maybe used in the methods of the invention is an α-anomeric nucleic acidmolecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual, 8-units, the strands run parallel to each other (Gaultier et al.(1987) Nucleic Acids. Res. 15:6625-6641; incorporated herein byreference). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148; incorporated herein by reference) or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBSLett. 215:327-330; incorporated hereinby reference).

In still another embodiment, an antisense nucleic acid molecule that maybe used in the methods of the invention is a ribozyme. Ribozymes arecatalytic RNA molecules with ribonuclease activity which are capable ofcleaving a single-stranded nucleic acid, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g. hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591;incorporated herein by reference)) can be used to catalytically cleavemRNA transcripts to thereby inhibit translation mRNAs. A ribozyme havingspecificity for an encoding nucleic acid molecule of interest can bedesigned based upon the nucleotide sequence of the cDNA. For example, aderivative of a Tetrahynena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in, an encoding mRNA of interest. See,e.g. Cech et al. U.S. Pat. No. 4,987,071; Cech et al. U.S. Pat. No.5,116,742; each of which is incorporated herein by reference.Alternatively, a mRNA of interest can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418;incorporated herein by reference.

In another embodiment, an agent that promotes RNAi can be used toinhibit expression of a negative regulator that counteracts the increasein expression and/or activity of an antioxidant defense protein(catalase, SOD2, PGC1α, and/or nrf2). RNA interference (RNAi is apost-transcriptional, targeted gene-silencing technique that usesdouble-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containingthe same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287.2431-2432 (2000); Zamore et al. Cell 101, 25-33 (2000). Tuschl et al.Genes Dev. 13. 3191-3197 (1999); Cottrell T R, and Doering T L. 2003.Trends Microbiol. 11:37-43; Bushman F.2003. Mol Therapy. 7:9-10; McManusM T and Sharp P A. 2002. Nat. Rev. Genet. 3:737-47; each of which isincorporated herein by reference). The process occurs when an endogenousribonuclease cleaves the longer dsRNA into shorter, e.g. 21- or22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. Thesmaller RNA segments then mediate the degradation of the target mRNA.Kits for synthesis of RNAi are commercially available from, e.g NewEngland Biolabsor Ambion. In one embodiment one or more of thechemistries described above for use in antisense RNA can be employed inmolecules that mediate RNAi.

Antibodies can also be used as agents in the methods of the invention.In one embodiment, an antibody is an intracellular antibody thatinhibits protein activity. Such an intracellular antibody is preparedusing methods well known in the art which generally involve preparing arecombinant expression vector which encodes the antibody chains in aform such that, upon introduction of the vector into a cell, theantibody chains are expressed as a functional antibody in anintracellular compartment of the cell.

For inhibition of transcription factor activity according to theinhibitory methods of the invention, an intracellular antibody thatspecifically binds the protein is expressed within the nucleus of thecell. Nuclear expression of an intracellular antibody can beaccomplished by removing from the antibody light and heavy chain genesthose nucleotide sequences that encode the N-terminal hydrophobic leadersequences and adding nucleotide sequences encoding a nuclearlocalization signal at either the N- or C-terminus of the light andheavy chain genes (see e.g. Biocca et al. (1990) EMBO J. 9:101-108;Mhashilkar et al. (1995) EMBO J. 14:1542-1551; each of which isincorporated herein by reference). A preferred nuclear localizationsignal to be used for nuclear targeting of the intracellular antibodychains is the nuclear localization signal of SV40 Large T antigen (seeBiocca et al. (1990) EMBO J. 9:101-108; Mhashilkar et al. (1995) EMBO J.14:1542-1551; each of which is incorporated herein by reference).

To prepare an intracellular antibody expression vector, antibody lightand heavy chain cDNAs encoding antibody chains specific for the targetprotein of interest, is isolated, typically from a hybridoma thatsecretes a monoclonal antibody specific for the protein. Antibodies canbe prepared by immunizing a suitable subject, (e.g. rabbit, goat, mouseor other mammal), e.g., with a protein immunogen. An appropriateimmunogenic preparation can contain, for example, recombinantlyexpressed protein or a chemically synthesized peptide. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory compound. Antibody-producingcells can be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975, Nature 256:495-497;incorporated herein by reference) (see also, Brown et al. (1981) J.Immunol 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh etal. (1976)PNAS 76:2927-31; Yeh et al. (1982)Int.J. Cancer 29:269-75;each of which is incorporated herein by reference). The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet., 3:231-36; each of which is incorporated herein byreference). Briefly, an immortal cell line (typically a myeloma) isfused to lymphocytes (typically splenocytes) from a mammal immunizedwith a protein immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds specifcally, aprotein of interest. Any of the many well known protocols used forfusing lymphocytes and immortalized cell lines can be applied for thepurpose of generating a monoclonal antibody (see, e.g. G. Galfre et al.(1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., citedsupra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, MonoclonalAntibodies, cited supra; each of which is incorporated herein byreference). Moreover, the ordinary skilled artisan will appreciate thatthere are many variations of such methods which also would be useful.

Typically, the immortal cell line (e.g. a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody thatspecifically binds the protein are identified by screening the hybridomaculture supernatants for such antibodies, e.g. using a standard ELISAassay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody that binds to a protein can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g. an antibody phage display library) with the protein, or a peptidethereof, to thereby isolate immunoglobulin library members that bindspecifically to the protein. Kits for generating and screening phagedisplay libraries are commercially available (e.g. the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurJZAP™ Phage Display Kit, Catalog No. 240612; each of whichis incorporated herein by reference).

Examples of methods and compounds particularly amenable for use ingenerating and screening antibody display libraries can also be foundin, for example, Ladner et al U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Grifeths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) JMol Biol 226:889-896;Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;Hoogenboom et al. (1991)NucAcid Res 19:4133-4137; Barbas et al. (1991)PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; each ofwhich is incorporated herein by reference.

In another embodiment, ribosomal display can be used to replacebacteriophage as the display platform for identifying antibodies for usein the methods of the invention (see, e.g. Hanes et al. 2000. Nat.Biotechnol. 18:1287; Wilson et al. 2001. Proc. Natl. Acad. Sci. USA98:3750; Irving et al. 2001 J. Immunol. Methods 248:31; each of which isincorporated herein by reference). In yet another embodiment, cellsurface libraries can be screened for antibodies (Boder et al. 2000.Proc. Natl. Acad. Sci. USA 97: 10701; Daugherty et al. 2000 J. Immunol.Methods 243:211; each of which is incorporated herein by reference).Such procedures provide alternatives to traditional hybridoma techniquesfor the isolation and subsequent cloning of monoclonal antibodies.

In another embodiment, an antibody that may be used in the methods ofthe invention is a substantially human antibody generated in transgenicanimals (e.g., mice) that are incapable of endogenous immunoglobulinproduction (see e.g. U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and5, 589,369 each of which is incorporated herein by reference).

For example, it has been described that the homozygous deletion of theantibody heavy-chain joining region in chimeric and germ-line mutantmice results in complete inhibition of endogenous antibody production.Transfer of a human immunoglobulin gene array to such germ line mutantmice will result in the production of human antibodies upon antigenchallenge. Another preferred means of generating human antibodies usingSCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporatedherein by reference. It will be appreciated that the genetic materialassociated with these human antibodies can also be isolated andmanipulated as described herein.

Yet another highly efficient means for generating recombinant antibodiesfor use in the methods of the invention is disclosed by Newman,Biotechnology, 10:1455-1460 (1992); incorporated herein by reference.Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in U.S. Pat. Nos.5,658,570, 5,693,780 and 5, 756,096; each of which is incorporatedherein by reference.

Once a monoclonal antibody has been identified (e.g. either ahybridoma-derived monoclonal antibody or a recombinant antibody from acombinatorial library, including monoclonal antibodies that are alreadyknown in the art), DNAs encoding the light and heavy chains of themonoclonal antibody are isolated by standard molecular biologytechniques. For hybridoma derived antibodies, light and heavy chaincDNAs can be obtained, for example, by PCR amplification or cDNA libraryscreening. For recombinant antibodies, such as from a phage displaylibrary, cDNA encoding the light and heavy chains can be recovered fromthe display package (e.g. phage) isolated during the library screeningprocess. Nucleotide sequences of antibody light and heavy chain genesfrom which PCR primers or cDNA library probes can be prepared are knownin the art. For example, many such sequences are disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U. S. Department of Health and Human Services, NIH PublicationNo. 91-3242 and in the “Vbase” human germline sequence database.

Once obtained, the antibody light and heavy chain sequences are clonedinto a recombinant expression vector using standard methods. Asdiscussed above, the sequences encoding the hydrophobic leaders of thelight and heavy chains are removed and sequences encoding a nuclearlocalization signal (e.g., from SV40 Large T antigen) are linkedin-frame to sequences encoding either the amino- or carboxy terminus ofboth the light and heavy chains. The expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly.

In another embodiment, an inhibitory agent for use in the methods of theinvention is a peptidic compound derived from the amino acid sequence ofa negative regulator that counteracts the increase in expression and/oractivity of an antioxidant defense protein (catalase, SOD2, PGC1α,and/or nrf2).

Peptidic compounds useful in the method of the invention can be madeintracellularly by introducing into the cells an expression vectorencoding the peptide. Such expression vectors can be made by standardtechniques using oligonucleotides that encode the amino acid sequence ofthe peptidic compound. The peptide can be expressed in intracellularlyas a fusion with another protein or peptide (e.g., a GST fusion).Alternative to recombinant synthesis of the peptides in the cells, thepeptides can be made by chemical synthesis using standard peptidesynthesis techniques.

Synthesized peptides can then be introduced into cells by a variety ofmeans known in the art for introducing peptides into cells (e.g.,liposome and the like).

Another form of an inhibitory agent which inhibits a negative regulatorthat counteracts the increase in expression and/or activity of anantioxidant defense protein (catalase, SOD2, PGC1α, and/or nrf2) in acell is a chemical small molecule compound.

Pharmaceutical Compositions of the Invention

In one aspect of the invention, a therapeutic nucleic acid moleculeand/or vector containing the same will be in the form of apharmaceutical composition containing a pharmaceutically acceptablecarrier. As used herein “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. In one embodiment, thepharmaceutically acceptable carrier is not phosphate buffered saline(PBS). In one embodiment, the carrier is suitable for intraocular,topical, parenteral, intravenous, intraperitoneal, or intramuscularadministration. In another embodiment, the carrier is suitable for oraladministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the gene therapy vector, use thereofin the pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In a particular embodiment, the pharmaceutical compositions of thepresent invention would be administered in the form of injectablecompositions. The compositions can be prepared as an injectable, eitheras liquid solutions or suspensions. The preparation may also beemulsified. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the preparation may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH-bufferingagents, adjuvants or immunopotentiators.

In a particular embodiment, the nucleic acid molecules and/or vectorsare incorporated in a composition suitable for intraocularadministration. For example, the compositions may be designed forintravitreal, subretinal, subconjuctival, sub-tenon, periocular,retrobulbar, suprachoroidal, and/or intrascleral administration, forexample, by injection, to effectively treat the retinal disorder.Additionally, a sutured or refillable dome can be placed over theadministration site to prevent or to reduce “wash out”, leaching and/ordiffusion of the active agent in a non-preferred direction.

Relatively high viscosity compositions, as described herein, may be usedto provide effective, and preferably substantially long-lasting deliveryof the nucleic acid molecules and/or vectors, for example, by injectionto the posterior segment of the eye. A viscosity inducing agent canserve to maintain the nucleic acid molecules and/or vectors in adesirable suspension form, thereby preventing deposition of thecomposition in the bottom surface of the eye. Such compositions can beprepared as described in U.S. Pat. No. 5,292,724, the entire contents ofwhich are hereby incorporated herein by reference.

Sterile injectable solutions can be prepared by incorporating thecompositions of the invention in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: A binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic, acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant: such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the compositions of the invention are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Nasal compositions generally include nasal sprays and inhalants. Nasalsprays and inhalants can contain one or more active components andexcipients such as preservatives, viscosity modifiers, emulsifiers,buffering agents and the like. Nasal sprays may be applied to the nasalcavity for local and/or systemic use. Nasal sprays may be dispensed by anon-pressurized dispenser suitable for delivery of a metered dose of theactive component. Nasal inhalants are intended for delivery to the lungsby oral inhalation for local and/or systemic use. Nasal inhalants may bedispensed by a closed container system for delivery of a metered dose ofone or more active components.

In one embodiment, nasal inhalants are used with an aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A non-aqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers maybe used to minimize exposing the agent to shear, which can result indegradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compositions of the invention can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of nucleic acid molecules describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds which exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

Data obtained from cell culture assays and/or animal studies can be usedin formulating a range of dosage for use in humans. The dosage typicallywill lie within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are hereby incorporated byreference.

EXAMPLES Materials and Methods Mouse Strains

Three mouse models of retinitis pigmentosa (RP), rd1, rd10 and rhodopsinknock-out (rho−/−), were chosen for the following studies. The rd1allele on the FVB/NJ background was chosen since these animals showrapid rod death, followed by relatively rapid cone death, due tohomozygosity of the phosphodiesterase 6 beta gene rd1 allele. Comparedto rd1, the phenotype of mice carrying the phosphodiesterase 6 beta generd10 allele is characterized by a slightly later onset of retinaldegeneration. The rd10 mice used in these studies were on a congenicC57BL/6J background and are, thus, suitable for behavioral assays andERG assays.

A third strain of mice used in these studies carries the rhodopsin rhonull allele (rho) which is described by Lem et al. (PNAS 96(2):736-41,1999). This strain has an intermediate speed of degeneration. Inaddition, mutation of the rhodopsin gene is the most common mutationfound in autosomal RP in humans.

Infection Protocol

Generally, P0 neonates were anesthetized on ice. About 0.3 μl of an AAVvirus was introduced into the subretinal space using a pulled glasspipette. Animals were allowed to survive for one week to one year, asseveral of the animal models for blindness have a slow degeneration.During this time, they were tested for visually guided behavior.Finally, they were euthanized and the histology of their eyes examined.

Because some of the experimental vectors do not encode a histologicalmarker, the vectors were co-injected with an equal titer of AAV-CMV-GFPto enable tracing of the infection with the use of the GFP. Controlvectors, AAV-CMV-GFP plus AAV-CMV-tdTomato, at the same final titer asthe experimental virus injections, were also injected in these cases. Acombination of GFP and tdTomato viruses were injected and it was shownthat 100% of cones were co-infected, and 85% of all cells in the ONLwere co-infected, which is primarily the co-infection rate for rods(FIG. 7). Injections into the sub-retinal space at P0 were made with 0.3microliters of virus suspension using a pulled glass pipette andEppendorf picospritzer. Animals were weaned and carried on standard chowand 12 hours light/12 hours dark until the assays described below wereperformed. The time points for analysis of rd1 mice were 8 weeks (W) and12 W. These time points were chosen based upon the previous descriptionsof the kinetics of cone death in rd1 mice, and are the points whereapproximately no cones remain in the central 50% of the retina (8W) andno cones within approximately 90% of the retina at 12 W.

Tests of Expression and Function

To initially test the vectors for protein expression and function, theywere assayed on wild-type (WT) retinas. P0 WT mice were co-infected withan equal ratio of AAV-CMV-GFP and an antioxidation vector, andsacrificed at P21. AAV8 is able to express within approximately oneweek, and most of the infected cells will be rods. The CMV promoter isnot as strong in rods as it is in cones, but it is strong enough to seeGFP expression immunohistochemically (see, e.g., FIG. 1E), as well as onWestern blots. Extracts were prepared for protein analysis, and aWestern blot was performed. Commercially available antisera was used(see, e.g., FIG. 2). Controls retinas were infected with only theAAV-CMV-GFP+/−AAV-CMV-tdtomato viruses. To assess how well the vector isexpressing, the level of the AAV transduced gene relative to GFP, andrelative to the endogenous level of the anti-oxidation gene, wasassessed. Since rods are 70% of the cells in the retina, this level waseasily visible on the blot.

Infected mice were assessed for the function of the anti-oxidation genesby assessment for oxidization using immunohistochemical detection ofacrolein on tissue sections. Sections were processed for acroleinstaining, as shown in FIG. 3.

Electroretinography (ERG)

Photopic ERG was conducted as per Komeima et al. (PNAS,103(30):11300-05, 2006). Generally, rd10 mice with higher b-wave arecorrelated with greater cone survival.

Mice were dark-adapted overnight and anesthetized, with both pupilsdilated. Rod-dominated responses were elicited in the dark with 10-msecflashes of white light (1.37×10⁵ cd/m²) presented at intervals of 1minute in a Ganzfeld dome. Light-adapted, cone responses were elicitedin the presence of a 41-cd/m² rod-desensitizing white background withthe same flashes (1.37×10⁵ cd/m²) presented at 1 Hz. ERGs were monitoredsimultaneously from both eyes, with signal averaging for cone responses.

Behavioral Assays

Behavioral assays include locomotion tests in light and darkenvironments (see, Collins et al., J of Neurosci Methods, 60(1-2):95-98, 1995; Legali et al., Nat Neurosci, 11(6):667-75, 2008),forced-choice swim test (see, Prusky et al., Vision Res, 40(16):2201-09,2000; Wong and Brown, Genes, Brain & Behav 5(5):389-403, 2006), anoptomotor test (see, Abdeljalil et al., Vision Res, 45(11):1439-46,2005; Legali et al., Nat Neurosci, 11(6):667-75, 2008; Prusky et al.,Invest Ophthal and Visual Sci, 45(12):4611-16, 2004), and a visual clifftest (see, Nagar et al., Neurosci, 160(2):517-29, 2009.

The Optomotor device looks like a rotating drum, with a platform in thecenter. Awake mice were placed on the platform and allowed to habituateto the chamber. This was assessed by whether they jump off of theplatform. It typically takes about 5 minutes to habituate the animal tothe device. The investigator watched the animals and once they remain onthe platform for 1-2 minutes, testing began. To simulate motion, stripeswere projected, flashing on and off, onto the inside surface of thedrum. The thickness of the stripe, frequency of flashes, contrast, andcolor were altered over time to determine the visual performance. Avideo camera was trained onto the mouse to evaluate its response to thesimulated movement. Blind mice did not respond; sighted mice increasedtheir body movements, and also turned their heads as if trackingmovement. Each mouse was tested a total of 4-6 times over the course ofan experiment, which lasted 3 weeks to 6 months. The optomotor test hasthe advantage that one can measure acuity and sensitivity, which are noteasily measured in the other tests. At the termination of theexperiment, the animals were tested for ERG prior to sacrifice.

Light-Evoked Ganglion Cell Activity Recording

Animals were dark-adapted over night before being anesthetized. Underinfrared illumination to minimize visible light exposure, retinas weredissected and mounted ganglion cell side up on a piece of filter paperin 37 degree oxygenated Ringer medium. The retinas were stimulated with1 second of visible light (365 nm+505 nm) at an intensity of 10¹⁰photons cm's⁻¹. Light-evoked spikes of a single ganglion cell wererecorded by an electrode, and responses were averaged by 20 trials.

Histological Analyses

Eyes were enucleated, placed in 4% formaldehyde in PBS, and theiranterior segments and lens were removed. Fixation continued in thisfixative for 30 minutes. The fixed tissues were soaked in 30%sucrose/PBS for 3 hours or overnight, shock frozen, and sectioned alongthe superior-inferior meridian at 20 μm thickness. Rhodamine-labeledpeanut agglutinin (PNA), red/green opsin, and blue opsin antibodies wereused for staining. Stained sections were photographed on a confocallaser scanning microscope. For quantitative comparison, the excitationenergy levels and duration of exposure were kept the same for pairedsamples, and images were taken to preserve the dynamic range of theoriginal signal intensities. Image data were analyzed quantitativelyusing the ImageJ and Imaris software.

The retinas were assessed histologically using two types of assays todetermine the number of cones. A flat mount of the entire retina wasgenerated and scored for the number of bright GFP cells in a consistentarea of each retina. Flat mount retinas were oriented based upon a markmade by a cauterizing iron at 12 o'clock, and 4 quadrants were markedoff. One square in each retinal quadrant was chosen for quantification,at a distance of 1.5 mm from the optic nerve head, on a line between theoptic nerve head and the periphery, as described by Komeima et al. (PNAS103(30):11300-05, 2006). 60× microscope fields were photographed in eachquadrant located at these distances, as exemplified in FIG. 1F. Thenumber of bright GFP+ cells was quantified using Imaris software.Retinal sections were also prepared and stained with anti-cone arrestinand/or PNA binding, along with anti-GFP (FIG. 1B). Sections were madealong the dorsoventral axis, in 50% of the retinas, and along theanterioposterior axis, in 50% of the retinas. Two sections near theoptic nerve head were quantified for the number of cells positive forthe cone marker throughout the entire section. Confocal imaging andImaris software was used for this analysis. However, in degeneratingretinas, it has been found that very dysmorphic cones often aredifficult to quantify for cone arrestin and PNA. The bright GFP from theCMV promoter of the virus has provided a more robust way to score, whichis why we also stained with anti-GFP.

Example 1: Effects of Antioxidant Enzymes Superoxide Dismutase (SOD2)and Catalase on Cone Cell Death in a Mouse Model of Retinitis Pigmentosa

It is generally recognized that cones show signs of oxidation in RP. Forexample, cones survive longer in mice that have been supplied withanti-oxidants in the diet, or have been transgenically modified tooverexpress an antioxidant gene. In addition, it has been shown thatcatalase, delivered via an Adenovirus vector to RPE cells, protectedadjacent photoreceptors from light damage (see, Rex et al., Human GeneTherapy 15(10):960-67, 2004). AAV mediated delivery of anti-oxidantenzymes directly to cones might then protect cones, and in addition,even if all cones are not infected, there may be a benefit to nearbyuninfected cones.

Experiments were carried out in rd1 and rd10 null mice at least threetimes for each strain. These two strains, with different upstreammutations and different kinetics of degeneration, were tested in orderto give a greater chance of success, should there be anystrain-specific, negative responses. Any positive, reproduciblecombinations will be tested in AAV vectors with cone-specific promoters.

AAV Vectors Encoding Antioxidant Defense Genes

Exemplary AAV vectors encoding the antioxidant defense genes, SOD2 andcatalase, are depicted in FIG. 8. SOD2, GPX4, and catalase have beenimplicated in photoreceptor viability (see, e.g., Usui et al., MolTherap 17(5):778-86, 2009; Ueta et al., JBC 287(10):7675-82, 2012). Acatalase allele with the modifications per Usai et al. (Mol Therap17(5):778-86, 2009), replacing the peroxisome targeting sequence withthe mitochondrial targeting sequence from ornithine transcarbamylase hasbeen made. This allele was found to be effective, while the normalperoxisome targeted version was not.

The vectors were packaged in AAV capsid type 8, with a tyrosine mutationin the C-terminal tyrosine, which augments infectivity of the viruspreparation. Site-directed mutagenesis was used to create this capsidallele. Production and purification of such vectors were done using anIodixinol gradient centrifugation, followed by ion exchange columnpurification. The final titers range from 10¹²-10¹⁴ virus particle(vp)/ml and have excellent infectivity (FIGS. 1 and 7).

Results AAV Vectors

AAV vectors have been used successfully in patients with, e.g., Leber'scongenital amaurosis. Thus, these vectors were chosen for the deliveryof genes to RP retinas. The promoters that can be used with AAV must berelatively small, owing to a rather limited capacity of the virus ofabout 4.7 Kb of DNA. CMV, although a fairly broadly expressed promoter,is more active in cones than in rods (FIGS. 1B, E). In addition, CMV candrive high level expression in the RPE. If there are excess freeradicals in the RPE as well as in the retina, this might have thebenefit seen in a study in which Adenovirus was used to transduce theRPE with catalase (see, Rex et al., Human Gene Therapy 15(10):960-67,2004). Thus, CMV was chosen for the initial experiments. By injecting atP0 rather than older stages, it was found that virtually every cone isinfected throughout the retina, when the virus is accurately deliveredto the subretinal space, which occurs in about ⅔ of the animals (FIGS.1A-D). This wide dissemination of virus is likely due to 2 causes. Oneis that the P0 retina subretinal space is relatively open due to thelack of OS and RPE connections at this early stage before OS arepresent. The viral inoculum is thus free to diffuse from the inoculationsite. Furthermore, the cones are situated near the scleral side of thesubretinal space, and there is not a layer of OS and inner segments (IS)that might interfere with virus diffusion and entry into the cells. Theyare, thus, readily accessed by the viral particles. In fact, thisinjection protocol was found to be so robust, and the CMV-GFP so bright,that the number of bright GFP cells can be used as a proxy for conesurvival (FIG. 1F). This allowed a relatively rapid assessment of conesurvival following delivery of test genes. Such results were followed upby staining for the cone markers, cone arrestin or PNA, in a secondaryscreen (FIG. 1B).

Anti-Oxidation Enzymes

The expression of anti-oxidant enzymes in wild type (WT) and RP retinas(rd, generally) during the course of degeneration (FIG. 2) was examined.The conversion of free radicals of oxygen to H₂O₂ is carried out by theSOD enzymes, SOD1-3. Following this reaction, 14202 is converted towater and oxygen by catalase or glutathione peroxidase. As the electrontransport chain in mitochondria is a major source of free radicals inall cells, and photoreceptors are known to have one of the highestdensities of mitochondria of all cell types in their IS, it is nosurprise that high levels of GPX1 and SOD2 were found in the IS ofphotoreceptors (FIGS. 2A-D).

After making these observations, it was determined whether rodphotoreceptors relied on SOD2 or GPX1 for their survival. To this end,small hairpin (sh)RNA plasmids were delivered to WT retinas usingelectroporation at P0 (see, Matsuda and Cepko, PNAS 104(3):1027-32,2007). This method targets primarily mitotic progenitor cells, whichpass the plasmids on to the daughter cells, 80% of which are rods.(Cones are post-mitotic at this time and are not electroporatedeffectively.) The hairpin plasmids were co-electroporated with a plasmidexpressing GFP, under the ubiquitous CAG promoter. There was a higherlevel of oxidized lipids, as revealed by acrolein staining, in retinaswith SOD2 knock-down (FIG. 3). The rods electroporated by the GPX1 shRNAdied, as seen by TUNEL staining (FIG. 4). These data support thehypothesis that photoreceptor cells require robust anti-oxidationcapacity. It is interesting that death occurred very rapidly upon lossof a single anti-oxidation enzyme, suggesting that these enzymes are notredundant. This may be due to their different activities, as GPX4 ismore active in lipid oxidation while GPX1 is not known to have anyspecific molecules that it targets. Further, enzyme localization may beimportant as anti-oxidation enzymes can target different compartments,i.e. mitochondria, cytoplasm, or peroxisomes.

Thus, two sets of vectors were made. In one set, two anti-oxidationdefense genes were expressed from the same AAV vector. To track suchinfections, co-infection with an AAV-CMV-GFP was used. As shown in FIG.1, AAV-CMV-GFP is very bright in cones and allows quantification, and asshown in FIG. 7, co-infection rates are high. In a second set ofvectors, the two anti-oxidation defense genes were expressed from twodifferent AAV vectors, with GFP included in at least one of the vectors.The advantage of having the genes in two different vectors is that itenables testing of various combinations of anti-oxidation genes, andtesting of only single gene.

Data from co-infection of P0 rd1 retinas with AAV-CMV-SOD2 andAAV-CMV-catalase show greater cone survival at P50 in rd1 miceco-infected with these vectors (FIGS. 10 and 11). The number of brightGFP cells in the scleral portion of the retina in sections taken throughthe central portion of the retina was quantified. The number of brightGFP+ cells in the control was 259+/−113 cones/section, and the number inthe SOD2+catalase infected retinas was 342+/−29.3 cones/section.

Additionally, SOD2 and catalase co-overexpression in rd1 retinas showimproved cone survival. At all the time points examined (P30, P50, P60,and P70), the cone density in retinas overexpressing SOD2+catalase ishigher than that in retinas with GFP overexpression. At postnatal day50, the cone density in retina overexpressing

SOD2+catalase is approximately 1.5-fold higher (196+/−22.3 cones/0.0625mm² vs 133+/−60 cones/0.0625 mm², p value<0.02) (FIG. 11). Further, coneouter segments, where phototransduction is carried out and which is animportant index for cone function, were better preserved in rd retinaupon SOD2+catalase co-overexpression (FIG. 12). While cone outersegments are comprised in rd1 retinas at postnatal day 60 (FIG. 12B),they are present in most remaining cones in retinas with SOD2 andcatalase overexpression (FIG. 12C). Quantification of this phenotypeshowed a great increase in the percentage of cones with obvious PNAstaining in retinas with SOD2 and catalase overexpression (FIG. 12D).

Moreover, mice overexpressing both SOD2 and catalase exhibited betteroverall photoreceptor function as assessed by optomotor response (FIG.13) and light-evoked ganglion cell activity (FIG. 14). Shown byoptomotor assay, the injected eyes with AAV vectors encoding SOD2 andcatalase have higher visual acuity compared to both the uninjected eyesand the control injected eyes at postnatal days 40 and 50 (FIG. 13). Atpostnatal day 50, the difference of left and right eye acuity (right eyeacuity-left eye acuity) is statistically significant between the controlgroup and antioxidant AAV treated group (0.0044 cycle/degree vs 0.0834cycle/degree, P<0.02) (FIG. 13B). In addition, retinas overexpressingSOD2 and catalase had increased light-evoked activity of ganglion cells(FIG. 14, left-hand side). Light response of surviving cones wasassessed by ganglion cell activity, which is the output signal fromretina to brain. The parameters (wavelength and intensity) of lightstimulus were selected to activate cone photoreceptors but notintrinsically photosensitive retinal ganglion cells (ipRGCs). Theaverage ganglion cell activity, measured by peak firing rate(spikes/second), was higher in retinas with SOD2 and catalaseoverexpression than in control retinas (106 spikes/sec vs 38.7spikes/sec, p<0.005) (FIG. 14). The mice that received antioxidant genetherapy will be further tested through other functional assays,including light-dark box assay, visual cliff assay, andelectroretinography.

The ability of anti-oxidant treatment to rescue cones in other retinaldegeneration models was tested by treating six month old rho−/− mouseretinas with AAV-GFP+SOD2+Catalase. The results shown in FIG. 25demonstrate that anti-oxidant treatment can be used to treat multiplemutations that lead to blindness.

Example 2: Effects of Transcription Factors PGC1 a and Nrf2 on Cone CellDeath in a Mouse Model of Retinitis Pigmentosa

Alternatively, or in combination with expression of at least oneantioxidant enzyme, a general upregulation of the anti-oxidation programmay provide greater cone survival (e.g., by up-regulation of theexpression and/or activity of PGC1α and Nrf2). The expression of PGC1αwas examined using immunohistochemistry in WT retinas (FIG. 5). Theprotein was found at a fairly high level in all retinal neurons, with ahigher level seen in cones than in rods.

An alternative master regulator is Nrf2, a basic leucin zippertranscription factor. Nrf2 is part of the endogenous cellular stressdefense mechanism and also has been shown to regulate transcription ofanti-oxidation enzymes. Nrf2 is usually kept at low levels in healthycells by its cytoplasmic binding partner Kelch-like ECH-associatedprotein 1 (Keap1) protein via ubiquitin-dependent degradation. Uponactivation by oxidants, Keap1-mediated Nrf2 turnover is disrupted, andNrf2 accumulates and translocates to the nucleus, where it modulates thetranscription of antioxidant genes with small Maf proteins through theantioxidant response element (ARE). The ARE is a cis-acting enhancerfound in the 5′ region of many antioxidant genes.

The cDNA for the mouse PGC1α allele (2.4 Kb) was inserted into theAAV2/8 vector system, allowing for expression of the PGC1α protein underthe control of a CMV promoter. PGC1α expression from AAV-CMV-PGC1α wasassayed using both Western blots of transfected, cultured cell lines,and by immunohistochemistry on infected retinas upon infection of Bl6Jretinas at P0. To determine PGC1α functionality, the expression level ofsome of its known target genes was assayed using semi-quantitativeRT/PCR. Briefly, retinal RNA preparations at P21 are obtained usingstandard methods (RNAeasy kit, Qiagen) and the levels of SOD2, GPX1 and4, catalase and uncoupling protein (UCP) are compared betweencontrol-infected and PGC1α-infected retinas. Assays for greateroxidation protection (acrolein and ELISA for carbonyl adducts) are alsoconducted, as described above, following challenge by paraquat.

The full-length allele of the mouse Nrf2 gene was obtained from Addgene.An AAV2/8 vector expressing the Nrf2 protein driven by the CMV promoterwas produced. Its expression was confirmed by Western blot on culturedcell lines transfected with AAV-CMV-Nrf2 plasmid, followed byimmunohistochemistry on infected retinas. RT/PCR for several targetgenes was carried out as described above.

Results

PGC1α protein is expressed in most retinal cell types, with a higherlevel in cone photoreceptors than rods (FIG. 5). As PGC1α positivelyregulates the mitogenic and antioxidant pathways, its expression patternindicates a higher metabolic demand and a requirement for greaterantioxidant capacity of cones. Nrf2, on the other hand, is expressed ata very low level in photoreceptors and other retinal cells in normalretinas (FIG. 6A). However, during the rod photoreceptor cell deathphase of the retinal degeneration, the Nrf2 protein level was elevatedas assessed by immunohistochemistry (FIGS. 6B-6C). As described before,the Nrf2-Keap1 system functions as a cellular sensor for oxidants. Thus,elevated Nrf2 levels suggest higher oxidative stress and a greater needto cope with oxidants in cones during rod degeneration. This increase ofNrf2 protein level in cones during retinal degeneration was observed intwo different rd mouse models with distinct genetic mutations,suggesting that increasing oxidative stress in cones is a generalproblem for retinal degeneration diseases, including retinitispigmentosa.

Co-overexpression of PGC1α and Nrf2 by AAV vectors prolongs conesurvival in rd1 retinas at P50 (FIG. 15). At P50, most of the conephotoreceptors that have died are in the central area of retinas, as thedegeneration progresses from the center to the periphery in rd mouseretinas (FIG. 15A). Overexpression of PGC1α and Nrf2 rescued conesurvival in the central retina at P50 (FIG. 15B).

Quantification of the rescue phenotype showed that PGC1α and Nrf2overexpression rescued cone survival, with a significantly higher conedensity in treated retinas than control retinas (273+/−6 cones/0.0625mm² vs 133+/−60 cones/0.0625 mm², p<0.05) (FIG. 16).

In addition, PGC1α and Nrf2 co-overexpression promoted cone survivalbetter than co-overexpression of SOD2 and catalase (p<0.001) at P50.Furthermore, this rescue remained significantly higher than that of thecontrol retinas (p<0.05) at P80 (FIG. 16).

Data from co-infection of P30 rd1 retinas transfected with AAV-CMV-PGC1αand AAV-CMV-Nrf2 show that PGC1α and Nrf2 co-overexpression preservescone outer segments in rd1 retinas at P30 (FIGS. 17 and 18), P40 andP50. Quantification of the cone outer segments showed that Nrf2overexpression preserved cone outer segments, with a significantlyhigher percentage in treated retinas than control retinas (p<0.05) (FIG.20).

Additionally, overexpression of Nrf2 alone, prolonged cone survival inrd1 retinas at P50 (FIG. 19). At P50, the mean cone density in retinasoverexpressing Nrf2 was higher than that in retinas with either PGC1αalone or with Nrf2 and PGC1α. Moreover, treatment of Nrf2 showed thatsuperoxide levels were reduced upon addition of Nrf2 to rd10 mice at P45(FIG. 21), and overexpression of Nrf2 reduced lipid oxidation in conesin rd1 mice at P30 (FIG. 22).

Furthermore, mice overexpressing Nrf2 were shown to exhibit betteroverall photoreceptor function as assessed by optomotor assay (FIG. 23),electroretinography (FIG. 24) and light-evoked ganglion cell activity(FIG. 14, right-hand side). Shown by optomotor assay, the injected eyeswith AAV vector encoding Nfr2 have higher visual acuity compared to boththe uninjected eyes and the control injected eyes at P50 (FIG. 23). Theratio of Right eye/Left eye visual acuity of each animal was used tominimize the variation between animals without treatment. The R/L ratioof AAV-CMV-Nrf2 treated retinas was significantly higher than that ofthe control treated retinas (p<0.05). In addition, retinasoverexpressing Nrf2 had a substantially better waveform as shown byelectroretinography analysis. The ratio of right eye/left eye b-waveamplitude was significantly higher in Nrf2 treated mice than that in thecontrol mice (FIG. 24).

Retinas overexpressing Nrf2 also had increased light-evoked activity ofganglion cells (FIG. 14). Light response of surviving cones was assessedby ganglion cell activity as described above. The average ganglion cellactivity was higher in retinas with Nrf2 overexpression than in controlretinas (FIG. 14). Results from these functional assays demonstrate thatcone function in mouse models of retinitis pigmentosa was preserved uponNrf2 treatment.

Example 3: Effect of the Transcription Factor Nrf2 and the AntioxidantEnzyme Superoxide Dismutase (SOD2) on Retinal Ganglion Cells

The retina is a structure with one white matter tract, the optic nerve,connecting the retinal ganglion cells (RGCs) to their targets within thebrain. The isolation of these axons from surrounding gray matterprovides a unique opportunity to create a pure axonal injury by crushingthe nerve to use as a model for glaucoma (see, e.g., Templeton andGeisert (2012) Mol Vision 18:2147-2152). Optic nerve crush (ONC) hasadvantages over other methods, such as optic nerve transections, for itis relatively mild and does not interrupt ocular blood flow. The ONC isparticularly useful as a simple synchronous approach for examiningganglion cell injury in a large number of mouse strains. Thisexperimental model produces an insult with the same molecular changesthat occur in murine models of glaucoma where there is both an inducedand/or intrinsic elevation of intraocular pressure.

Accordingly, the effect of increased expression of the antioxidantdefense proteins, Nrf2 and Sod2, on the survival of RGCs following opticnerve crush, was examined. Wild-type mice whose one eye was infectedwith AAV2-GFP (n=4), AAV2-Nrf2 (n=8), or AAV2-Sod2 (n=8) two weeksprior, were anesthetized and the optic nerve was crushed. Two weeksafter crush, the animals were sacrificed, flat mounts of the retinaswere prepared, and the number of RGCs were counted byimmunohistochemical staining with an RGC marker, neuronal betaIII/tubulin marker TUJ1, and an axonal regeneration marker, GAP43. Asdemonstrated in FIG. 26, although treatment with Nrf2 or Sod2 did notincrease axon regeneration (FIG. 26B), both Nrf2 and Sod2 significantlypromoted RGC survival, as compared to treatment with AAV2-GFP (FIG.26A). Accordingly increased expression of one or more antioxidantdefense proteins is useful for treating a retinal disorder, such asglaucoma in a subject.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method for treating or preventing retinitis pigmentosa in a subject, comprising administering to the subject a pharmaceutical composition formulated for intraocular administration, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) comprising a promoter operably linked to a nucleic acid molecule encoding nuclear factor erythroid 2-like 2 (Nrf2) in an amount effective for promoting photoreceptor survival and/or function and a pharmaceutically acceptable carrier for intraocular administration, wherein the promoter is a retinal pigment epithelial-specific promoter, a rod-specific promoter, a cone-specific promoter, or a rod- and cone-specific promoter, wherein the recombinant AAV is an AAV8 or AAV 2/8 recombinant AAV, and wherein the pharmaceutical composition is formulated for intraocular administration, thereby treating or preventing retinitis pigmentosa in the subject.
 2. The method of claim 1, wherein the promoter is selected from the group consisting of an Nrl promoter, a Crx promoter, a Rax promoter, a cone opsin promoter, an interphotoreceptor retinoid binding protein (IRBP156) promoter, a rhodopsin kinase (RK) promoter, a neural leucine zipper (NRLL) promoter, a cone arrestin promoter, a Cabp5 promoter, a Cralbp promoter, and combinations thereof.
 3. The method of claim 1, wherein the pharmaceutical composition further comprises a viscosity inducing agent.
 4. The method of claim 1, wherein the intraocular administration is selected from the group consisting of intravitreal, subretinal, subconjunctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and intrascleral administration.
 5. A method for treating or preventing retinitis pigmentosa in a subject, comprising administering to the subject a pharmaceutical composition formulated for intraocular administration, wherein the pharmaceutical composition comprises an adeno-associated virus (AAV) comprising a retinal pigment epithelial-specific promoter, a rod-specific promoter, a cone-specific promoter, or a rod- and cone-specific promoter operably linked to a nucleic acid molecule encoding nuclear factor erythroid 2-like 2 (Nrf2) in an amount effective for prolonging the viability of a photoreceptor cell, and a pharmaceutically acceptable carrier for intraocular administration, wherein the pharmaceutical composition is formulated for intraocular administration, thereby treating or preventing retinitis pigmentosa in the subject.
 6. The method of claim 5, wherein the AAV vector is an AAV 2/5 or an AAV 2/8 vector.
 7. The method of claim 5, wherein the promoter is selected from the group consisting of an Nrl promoter, a Crx promoter, a Rax promoter, a cone opsin promoter, an interphotoreceptor retinoid binding protein (IRBP 156) promoter, a rhodopsin kinase (RK) promoter, a neural leucine zipper (NRLL) promoter, a cone arrestin promoter, a Cabp5 promoter, a Cralbp promoter, and combinations thereof.
 8. The method of claim 5, wherein the pharmaceutical composition further comprises a viscosity inducing agent.
 9. The method of claim 5, wherein the intraocular administration is selected from the group consisting of intravitreal, subretinal, subconjunctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and intrascleral administration.
 10. A method for prolonging the viability of a photoreceptor cell compromised by retinitis pigmentosa in a subject, comprising administering to the subject a pharmaceutical composition formulated for intraocular administration, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) comprising a promoter operably linked to a nucleic acid molecule encoding nuclear factor erythroid 2-like 2 (Nrf2) in an amount effective for promoting photoreceptor survival and/or function and a pharmaceutically acceptable carrier for intraocular administration, wherein the promoter is a retinal pigment epithelial-specific promoter, a rod-specific promoter, a cone-specific promoter, or a rod- and cone-specific promoter, wherein the recombinant AAV is an AAV8 or AAV 2/8 recombinant AAV, and wherein the pharmaceutical composition is formulated for intraocular administration, thereby prolonging the viability of a photoreceptor cell compromised by retinitis pigmentosa in the subject.
 11. The method of claim 10, wherein the promoter is selected from the group consisting of an Nrl promoter, a Crx promoter, a Rax promoter, a cone opsin promoter, an interphotoreceptor retinoid binding protein (IRBP 156) promoter, a rhodopsin kinase (RK) promoter, a neural leucine zipper (NRLL) promoter, a cone arrestin promoter, a Cabp5 promoter, a Cralbp promoter, and combinations thereof.
 12. The method of claim 10, wherein the pharmaceutical composition further comprises a viscosity inducing agent.
 13. The method of claim 10, wherein the intraocular administration is selected from the group consisting of intravitreal, subretinal, subconjunctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and intrascleral administration.
 14. A method for prolonging the viability of a photoreceptor cell compromised by retinitis pigmentosa in a subject, comprising administering to the subject a pharmaceutical composition formulated for intraocular administration, wherein the pharmaceutical composition comprises an adeno-associated virus (AAV) comprising a retinal pigment epithelial-specific promoter, a rod-specific promoter, a cone-specific promoter, or a rod- and cone-specific promoter operably linked to a nucleic acid molecule encoding nuclear factor erythroid 2-like 2 (Nrf2) in an amount effective for prolonging the viability of the photoreceptor cell, and a pharmaceutically acceptable carrier for intraocular administration, wherein the pharmaceutical composition is formulated for intraocular administration, thereby prolonging the viability of a photoreceptor cell compromised by retinitis pigmentosa in the subject.
 15. The method of claim 14, wherein the AAV vector is an AAV 2/5 or an AAV 2/8 vector.
 16. The method of claim 14, wherein the promoter is selected from the group consisting of an Nrl promoter, a Crx promoter, a Rax promoter, a cone opsin promoter, an interphotoreceptor retinoid binding protein (IRBP156) promoter, a rhodopsin kinase (RK) promoter, a neural leucine zipper (NRLL) promoter, a cone arrestin promoter, a Cabp5 promoter, a Cralbp promoter, and combinations thereof.
 17. The method of claim 14, wherein the pharmaceutical composition further comprises a viscosity inducing agent.
 18. The method of claim 14, wherein the intraocular administration is selected from the group consisting of intravitreal, subretinal, subconjunctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and intrascleral administration. 